Chimeric system for functionally expressing and screening catsper channel

Abstract

The present invention is directed to chimeric CatSper channel proteins which are engineered by replacing the cytoplasmic domain of CatSper with those from a related channel with proven surface localization. The novel chimeric channels with the intact CatSper pore successfully traffic to the surface of mammalian cells and conduct calcium. The invention therefore is directed to chimeric CatSper channel proteins, nucleotide sequences which encode for such channel proteins, expression vectors which express such proteins and mammalian cells in which CatSper channel proteins have localized onto cell surfaces as well as methods for recording calcium flux in cells which comprise chimeric CatSper channel proteins localized on the cell surface. The present invention can be used to identify ligands and molecules which exhibit inhibition of fertility (contraception) or enhanced fertility (fertility agents).

Description

[0001] Chimeric System for Functionally Expressing and Screening CatSper Channel

[0002] Field of the Invention

[0003] The present invention is directed to chimeric CatSper channel proteins which are engineered by replacing the cytoplasmic domain of CatSper with those from a related channel with proven surface localization. The novel chimeric channels with the intact CatSper pore successfully traffic to the surface of mammalian cells and conduct calcium. The invention therefore is directed to chimeric CatSper channel proteins, nucleotide sequences which encode for such channel proteins, expression vectors which express such proteins and mammalian cells in which CatSper channel proteins have localized onto cell surfaces as well as a method for imaging calcium flux in cells which comprise chimeric CatSper channel proteins localized on the cell surface. Other aspects, including additional embodiment of the present invention are described herein.

[0004] Related Applications and Grant Support

[0005] This application claims the benefit of priority of US provisional application serial number 63 / 729,537, filed 9 December 2024, the entire contents of said application being incorporated by reference herein.

[0006] This invention was made with government support under R61HD114344 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] Background and Overview of the Invention

[0008] Birth rates are decreasing as women increasingly are having smaller families. But even as the birth rate decreases, the percentage of those births that are unplanned or unwanted has remained a constant for the last 30 years. Globally, from 1992 to 2014, around 45% of pregnancies were unintended (Sedgh, Singh et al. 2014). Contraceptive failure is responsible for about half of unintended pregnancies (Bearak, Popinchalk et al. 2018). Contraceptive non-use, responsible for the other half of unintended pregnancies, is largely attributed to concerns about side effects (Mosher and Jones 2010). Female contraceptives have been available in various forms for over 60 years. By contrast, there are no reversible, non-barrier methods of contraception available to males, despite growing interest and acceptance from prospective users (Dorman and Bishai 2012, Bakkensen and Feinberg 2021). At least 17 million men in the US alone are looking for alternatives to condoms and vasectomy; an estimated market of $1 billion by 2024. These data indicate high demand for a novel contraceptive technology that is non-hormonal, reversible, free of side effects, and works in male subjects. The sperm and the eggs are two players in achieving fertilization. Thus, an ideal male contraceptive will not damage the sperm production capability and be reversible. Targeting a molecule that specifically works at post-testicular and pre-fertilization stages, such as blocking transporting sperm, sperm motility and / or the fertilizing capability, and will carry minimal off-target side effects is a goal of this invention.

[0009] At the same time, approximately 1 in 6 couples are affected by infertility (Mascarenhas, Cheung et al. 2012). Male factors account for around 50% of infertile couples (Agarwal, Mulgund et al. 2015). 90% of male infertility is caused by low sperm count, with poor sperm motility been also an important factor. Currently, there is no treatment to improve sperm motility, which leads to women principally carrying the burden. Options for couples with male infertility due to low count or poor sperm motility are expensive and limited to hormonal treatments and assisted reproductive technologies (ART) such as intrauterine insemination (IUI) and in vitro fertilization (IVF) which often requires intracytoplasmic sperm injection (ICSI). The global male fertility market size is expected to reach of $5 billion by 2027. Thus, identifying molecules that can improve sperm motility will increase knowledge of fertility and improve male fertility by enhancing natural conception and ART effectiveness in vitro.

[0010] Two crucial changes that occur during capacitation represent a motility change (i.e. the sperm flagellum beats vigorously and asymmetrically, producing a whip-like motion) and the acrosome reaction (i.e. an exocytotic event in the sperm head). The motility pattern -known as hyperactivated motility - enables the sperm to reach the egg by overcoming the viscous microenvironment of the female reproductive tract. Additionally, hyperactivation allows sperm to push through the sticky egg coat, and eventually fertilize the egg.

[0011] Maintaining sperm motility over time in vitro (Qi, Moran et al. 2007, Hwang, Mannowetz et al. 2019, Hwang, Wang et al. 2021), developing hyperactivation and triggering acrosome reaction all require an elevation of intraflagellar calcium entering through the CatSper channel and its downstream Ca signaling (Chung, Miki et al. 2017, Ded, Hwang et al. 2020). Thus, the CatSper channel is indispensable for the process of sperm capacitation and, consequently, for male fertility.

[0012] Ion channels and membrane receptors are major targets for drug molecules, as they are localized in the cell membrane and highly accessible to drugs. Indeed, over 20% of drugs approved for human diseases target ion channels (Kaczorowski, McManus et al. 2008, Santos, Ursu et al. 2017). Thus, ligands or drugs that modulate ion channel activity critical for sperm function and male fertility, but not for spermatogenesis, are useful in both intervening fertility and infertility treatment in males and will overall contribute to both men’s and women’s reproductive health. Since the first discovery of the sperm-specific and Ca -selective CatSper channel in 2001 (Ren, Navarro et al. 2001, Carlson, Westenbroek et al. 2003), considerable interest and efforts have been focused on this channel because it meets all the criteria for developing male contraceptives and also has great potential for improving natural conception and developing sperm-based ART enhancers as loss of CatSper function is sufficient to compromise fertilizing capacity in both mouse and human.

[0013] Freshly ejaculated mammalian sperm must undergo a physiological process called capacitation to be capable of fertilizing the egg. Two crucial changes that occur during capacitation represents a motility change (i.e. the sperm flagellum beats vigorously and asymmetrically, producing a whip-like motion) and the acrosome reaction (z.e. an exocytotic event in the sperm head). The motility pattern - known as hyperactivated motility - enables the sperm to reach the egg by overcoming the viscous microenvironment of the female reproductive tract. Additionally, hyperactivation allows sperm to push through the sticky egg coat, and eventually fertilize the egg. Maintaining sperm motility over time in vitro (Qi, Moran et al. 2007, Hwang, Mannowetz et al. 2019, Hwang, Wang et al. 2021), developing hyperactivation and triggering acrosome reaction all require the elevation of the intraflagellar calcium entered through the CatSper channel and its downstream Ca signaling (Chung, Miki et al. 2017, Ded, Hwang et al. 2020). Thus, CatSper channel is absolutely required for sperm capacitation process and male fertility.

[0014] CatSper is the most complex ion channel known, with at least thirteen proteins: four subunits that form a heterotetrameric channel (CATSPERl-4)(Ren, Navarro et al. 2001, Qi, Moran et al. 2007), as well as nine additional, non -pore forming subunits, including four single-pass transmembrane (TM) proteins with large extracellular domains (ECD) (CATSPERP, y, 6, and a) (Liu, Xia et al. 2007, Wang, Liu et al. 2009, Chung, Navarro et al.

[0015] 2011, Chung, Miki et al. 2017), two multi-pass TM proteins with small ECD (Lin, Ke et al.

[0016] 2021), two smaller cytoplasmic, calmodulin (CaM)-IQ domain proteins that form the EFCAB9-CATSPER^ complex (Chung, Miki et al. 2017, Hwang, Mannowetz et al. 2019, Hwang, Maziarz et al. 2021) and the C2-domain containing CatSperr (Hwang, Wang et al.

[0017] 2021). Deletions or mutations of any of the pore-forming or other TM-subunits results in the loss of the entire CatSper channel complex (Wang, McGoldrick et al. 2021). Two very recent studies, including the inventor’s own work, reported first time CatSper structures resolved by cryo-electron microscopy: in-cell structure and high order arrangement in mouse and human sperm (Zhao, Wang et al. 2021) and atomic structure of purified mouse CatSper complex (Lin, Ke et al. 2021). The structures reveal that the pore-forming channel (CatSperl-2-3-4 in counterclockwise direction) is covered by a canopy formed by the ECDs of the TM auxiliary subunits. The new insights from these CatSper structures provide thrust for developing new strategies of specifically targeting the structurally exposed surface of the channel for design and development of modulatory compounds.

[0018] Loss of CatSper function through the absence of CatSper transmembrane (TM) subunits (CatSper 1, 2, 3, 4, 6 or s) causes male infertility in mice with no other phenotypes (Wang, McGoldrick et al. 2021). The knockout sperm cells fail to develop hyperactivated motility, the asymmetric beating of tail required for fertilization. In humans, homozygous deletions and / or mutations in CatSper 1, 2, and a genes were reported in sterile men (Avidan, Tamary et al. 2003, Avenarius, Hildebrand et al. 2009, Brown, Miller et al. 2018). Most of all, CatSper inhibition is expected to have minimal, if not non-existent, side effects. CatSper is specifically expressed in post-meiotic male germ cells (Chung, Navarro et al. 2011, Chung, Miki et al. 2017). CatSper subunits are not expressed in any cell type other than male germ cells. Even in male germ cells, CatSper genes are not expressed before meiosis and are not involved in normal sperm production (Wang, McGoldrick et al. 2021). Functional CatSper channels appear only in the flagellar membrane of sperm tail. Thus, drugs inhibiting or activating CatSper would work at post-testicular and pre-fertilization stages and are unlikely to have undesired off-target side effects in either men or women. Since the CatSper channel is localized in the flagellar membrane (Kirichok, Navarro et al. 2006, Chung, Shim et al. 2014), CatSper subunits are highly accessible and amenable to reverse the drug binding. Despite this promise, the CatSper channel has been intractable to direct, high-throughput drug screening. Many attempts to reconstitute the CatSper channel in vitro have failed because the recombinant pore-forming subunits (CatSper 1-4) are retained in the endoplasmic reticulum (ER) and Golgi apparatus, failing to traffic to the plasma membrane. The complexity and high order arrangement of the channels might be one of the reasons for this failure (Lin, Ke et al. 2021, Wang, McGoldrick et al. 2021, Zhao, Wang et al. 2022, Hwang and Chung 2023). Nevertheless, the successful use of a CATSPER1 peptide epitope as an immunocontraceptive(Li, Ding et al. 2012) provides proof-of-concept for targeting the CatSper pore-forming subunits. Thus, functionally characterizing this important channel in a controlled manner is much needed.

[0019] Pursuant to the present application, it is reported that replacing the soluble, cytoplasmic domains of human CatSper calcium channels with those of diatom encoded single-domain (ID), voltage-gated channels (EukCatAs)(Helliwell, Chrachri et al. 2019) overcomes ER / Golgi retention of CatSper channels, enabling the channel to traffic to the plasma membrane in mammalian cells. Calcium imaging studies in HEK293 cells expressing the chimeric EukCatA-huCatSper channels reveal that the chimeric channels form the functional calcium channel, allowing the influx of calcium. These diatom-based interspecies chimeric systems provide new methods for functionally expressing CatSper channel that have not been successful to express in mammalian cells, and potentially other likewise difficult-to-express membrane proteins, suitable for high throughput screening to identify compounds modulating the activities.

[0020] 2+ 2+

[0021] Ca signaling mediated by the sperm specific CatSper Ca channel is required for male fertility in mammals by regulating sperm motility endurance, hyperactivation, acrosome reaction and sperm navigation in the female reproductive tract. In humans, mutations in CatSper genes have been reported in infertile male patients (Wang, McGoldrick et al. 2021). Thus, CatSper channel is a validated target for a male contraceptive development and to enhance sperm motility in assisted reproductive technology. However, as discussed, CatSper channels have not been successful candidates for direct screening for compounds that modulate their activity due to an inability to achieve functional expression of the channels in mammalian systems suitable for high throughput screening. The sperm-specific CatSper calcium channel was not amenable for drug screening because reconstitution of the channel in heterologous cells was not achieved because CatSper does not reach the cell surface but remains stuck inside the cells (e.g., ER and Golgi) when expressed in non-sperm cells.

[0022] Pursuant to the present invention, CatSper chimeric channels are engineered by fusing the transmembrane domains of human CatSper and the soluble domains of single-domain cation channels from diatom species. The chimeric channels produced traffic to the plasma membrane and assemble functional channels that conduct calcium ions. These findings serve as a foundation for establishing platforms to screen for ligands and modulators that can bind to the transmembrane domains and the pore of the CatSper channel. The chimeric CatSper channels of the present invention may also be used to study mechanisms of action for the identified hits and lead compounds from screening.

[0023] There is a substantial commercial opportunity in the present invention. The US contraceptive market size is estimated to worth $10 billion by 2027. The market is mainly addressed to woman, with men lacking contraceptive options to equally participate in family planning. According to Global Market Insights “if a new male contraceptive method is approved in the next five years, the market is projected to be worth about $1 billion by 2024 and could grow at a rate of 6% over the next 10 years”. The described invention entails the first screening platform for the development of a male contraceptive inhibiting CatSper ion channel.

[0024] The US market for infertility drugs is expected to reach $1.3 billion in 2022.

[0025] Infertility affects 15% of couples, and male infertility accounts for 40% of the cases. 90% of male infertility is caused by low sperm count, with poor sperm motility been also an important factor. There are no treatments to improve sperm motility. Options for couples with male infertility due to low count or poor sperm motility are expensive and limited to intrauterine insemination (IUI), in vitro fertilization (IVF), or sperm donation. The proposed invention entails the first screening platform to identify activators of CatSper ion channel, which will result in improved sperm motility and egg fertilization.

[0026] All previous drug screening to find CatSper modulators were performed using human sperm cells, which are difficult to maintain stable supply, control lot-to-lot signal to noise, thus limiting the screening largely applicable to indirect screening without a method to establish a good negative control. A few attempts led to identify hits with micromolar affinity without understanding the mechanisms of action. Now with the cells expressing chimeric CatSper, this invention is positioned not only to be able to test new small molecules to identify new hits and further develop lead compounds, but also to test previously identified hits from other screen for their specificity. Also depending on the CatSper expression level of the cell lines, this invention has advantage to screen not only inhibitors but also activators, expanding the scope of application for both developing contraceptives and infertility treatment (as enhancer of assisted reproductive technologies).

[0027] Brief Description of the Invention

[0028] The present application is directed to the discovery that replacing the soluble, cytoplasmic domains of human CatSper calcium channels with those of cation channels well-expressed in heterologous systems overcomes ER / Golgi retention of CatSper channels, thus enabling the channel to traffic to the plasma membrane in mammalian cells. Calcium imaging studies and electrophysiological recordings in HEK293 cells expressing the chimeric human CatSper channels form a functional calcium channel that recapitulates key biophysical properties of the native channel. These chimeric systems provide new methods for functionally expressing CatSper channels that have not been successful previously to express in mammalian cells, as well as other difficult-to-express flagellar ion transporters. These systems are suitable for high throughput screening (HTS) to identify novel channel modulators.

[0029] In an embodiment, the present invention is directed to single domain (ID) chimeric CatSper polypeptide channels which are engineered by replacing the two cytoplasmic domains of CatSper 1, 2, 3 or 4 with two cytoplasmic domains from a related channel with proven surface localization. Each CatSper transmembrane domain comprises two physiologically distinctive regions, a voltage sensing domain (S1-S4) and a pore-forming region S5-S6. In embodiments, these replacement cytoplasmic domains are from diatoms, in particular O. sinensis and P. tricornatum. The resulting novel chimeric polypeptide channels with the intact CatSper pore-forming channel (S1-S6) and cyotoplasmic domain substitution set forth herein as SEQ ID Nos: 1-8 (Tablet, Example 1) successfully traffic to the surface and conduct calcium in mammalian cells, and in particular, COS7 and HEK293 cells, among other mammalian cells and be used to identify agents with potential as infertility (contraceptive) agents or fertility agents (to provide hyperactivated motility and enhanced acrosome activity) by measuring calcium flows in the cell and determining how an agent influences calcium flow.

[0030] In an embodiment, the present invention is also directed to a chimeric huCatSper channel which uses the Cav3.2 as a structural scaffold to generate huCatSper as a single polypeptide (hCav3.2-hCatSperl-4-3-2 channel) as identified in the native CatSper channel. This chimeric CatSper channel comprises six transmembranes of each of human CatSper 1-4 assembled into a single chain heterotetramer with a specific counterclockwise order (1 -4-3-2) of these CatSper subunits. These subunits are tandemly linked (fused) in their native 1-4-3-2 order by replacing the four six transmembrane domains of the human Cav3.2 channel with each of human CatSper 1-4, with the N-terminal and C-terminal soluble domains and the loops linking them from Cav3.2 at the end of each CatSper subunit. The resulting chimeric human CatSper channel traffics to the plasma membrane in mammalian systems. A representation of this chimeric peptide channel is shown in FIGURE 41A. The peptide sequence of chimeric CatSper channel hCav3.2-hCatSper-l-4-3-2, described above is shown in FIGURE 41B and in Table 2 (at the end of Example 2) as SEQ ID No:9, which are identical sequences.

[0031] In embodiments, the present invention is directed to chimeric human CatSper channel proteins according to the peptide sequences SEQ. ID NOs: 1-8 which appear in Table 1 in the present application. In embodiments, the chimeric CatSper channel proteins comprise human CatSper 1 channel protein, human CatSper 2 channel protein, human CatSper 3 channel protein or human CatSper 4 protein wherein the soluble, cytoplasmic domains of each of the aforementioned CatSper proteins 1-4 is replaced with cytoplasmic domains of diatom encoded single domain (ID), voltage-gated channels of Phaeodactylum tricornutum (PtEUKATAl) or Odontella sinesis (Os). In embodiments, the channel proteins also include a fluorescent protein such as EFGP or mCherry, among others. In embodiments, chimeric CatSper channel proteins comprise six transmembrane domains of human CatSper (1, 2, 3 or 4) and a N-terminal soluble domain (EukCatAl) and a C-terminal soluble domain (EukCatAl) of O. sinensis or P. tricornutum. Illustration of these chimeric channel proteins and their sequences are presented in enclosed FIGURES 17-23. In embodiments, the present invention is directed to human chimeric CatSper protein huCav3.2-huCatSper, described above, which is presented as SEQ ID NO:9 in Table 2 herein. Illustration of this human chimeric channel protein is presented in FIGURES 41 A and 41B.

[0032] In embodiments, the present invention is also directed to polynucleotides which encode the human chimeric CatSper channels (SEQ ID Nos: 1-9) and can be used to provide polynucleotide expression vectors according to the present invention. Exemplary polynucleotide vectors which express the human chimeric CatSper channels pursuant to the present invention include OsEukCatAl-hCatSperl (FIGURES 10 and 35), OsEukCatAl-hCatSper2 (FIGURE 11 and 36), OsEukCatAl-hCatSper3 (FIGURE 1 and 37), OsEulCatAl-hCatSper4 (FIGURE 12 and 31), PtEukCatAl-hCatSperl (FIGURE 13), PtEukCatAl-hCatSper2 (FIGURE 14), PtEukCatAl-hCatSper3 (FIGURE 15), PtEukCatAl-hCatSper4 (FIGURE 16) and hCav3.2-hCatSper (FIGURES 39 and 40).

[0033] Pursuant to the present invention polynucleotide vectors (e.g. plasmid phCMV3 of FIGURES 10, 35, 11, 36, 1, 37, 12, 31, 13, 14, 15, 16 and 39 and lentiviral vector of FIGURE 40) are engineered to encode and express the chimeric CatSper channel proteins in mammalian, e.g. COS7 and HEK293 cells and other cells in culture. The vectors are typically cloned and subsequently transfected into cells for expression. Typically, the transfected expression cells are transient, mammalian expression cells.

[0034] The cloned expression vectors typically are transfected into mammalian cells (COS7 or HEK293) and grown in culture where the proteins encoded by the plasmid are expressed. In embodiments, the polynucleotide vectors are plasmid vectors, for example, as represented by the plasmid vectors which are presented in FIGURES 1, 10-16, 35-37 and 39-41 which are transfected into mammalian cells. These vectors express the representative chimeric CatSper channel protein in the cells into which the plasmid has been transfected. In embodiments, the expression vectors are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, etc.). In embodiments, the expressed chimeric channel proteins become localized on the surface of the cells in which the plasmids have been transfected. In embodiments, the chimeric channel proteins include a fluorescent protein such as EFGP or mCheery as reporters. In embodiments, the cells which contain localized chimeric CatSper channel protein are used in applications which involve Ca imaging and / or electrophysiological recordings to measure calcium flux. In embodiments, the transfected cells are selected to establish stable, monoclonal mammalian cells. In embodiments, these cells are COS7 or HEK293 cells.

[0035] The cell lines derived from the chimeric constructs according to the present invention are the first cell lines to express functional (Ca conducting) human CatSper pore in any heterologous system, which will allow direct, robust, and reproducible drug screening and further mechanism of action studies.

[0036] In embodiments, the present invention is directed to a method of identifying a compound of unknown CatSper channel activity as an inhibitor or an activator / enhancer of CatSper activity, comprising providing stable, transfected cells which express a chimeric CatSper channel according to the present invention at the surface of the cells and determining the impact of exposure of said compound on calcium flow through the chimeric CatSper channels expressed by said cells, wherein a compound which is shown to inhibit calcium flow through the CatSper channel is identified as a potential contraceptive agent and wherein a compound which is shown to increase or enhance calcium flow through the CatSper channel is identified as a potential fertility enhancing agent. In embodiments, the method comprises comparing the inhibition or enhancement of calcium flow said CatSper channel with a standard and determining whether said compound is identified with potential contraceptive or fertility enhancement activity. In embodiments, the standard is a known inhibitor of CatSper channel calcium flow. In embodiments, the standard is a known fertility enhancer of CatSper channel calcium flow.

[0037] In embodiments, the present invention is directed to a method of identifying a compound of unknown CatSper channel activity as an inhibitor or an activator / enhancer of CatSper activity, comprising providing stable, transfected cells which express a chimeric CatSper channel according to the present invention at the surface of the cells, establishing a baseline, resting calcium flow through said channel and determining the impact of exposure of said compound on the calcium flow through the chimeric CatSper channels expressed by said cells, wherein a compound which is shown to inhibit calcium flow through the CatSper channel is identified as a potential contraceptive agent and wherein a compound which is shown to increase or enhance calcium flow through the CatSper channel is identified as a potential fertility enhancing agent. In embodiments, the results of calcium flow for a particular compound are compared with a standard, which could the results for a known inhibitor of CatSper calcium flow (potential contraceptive agent) or a known enhancer of CatSper calcium flow (potential fertility enhancing agent).

[0038] In embodiments directed to a method of identifying a compound of unknown CatSper channel activity as an activator of CatSper and consequently, a potential fertility (enhancer of CatSper channel activity) agent,

[0039] the method comprises the steps of plating a population of stable transfected cells according to the present invention and incubating these cells for about 18-30 hours, often 24 hours in cell media;

[0040] removing the cell media from the incubated cells and replacing the cell media with buffer solution (e.g. Hanks buffered saline solution HBSS);

[0041] adding calcium dye (BD calcium dye) to the cells and incubating the cells in the buffered saline solution for approximately 15 minutes to one hour, often 30 minutes at approximately body temperature (about 37°C) and then for approximately 15 minutes to one hour, often 30 minutes at room temperature;

[0042] providing a concentration of compounds of unknown activity which are diluted in buffer serum which includes DMSO (often 0.1%) as negative control (no activation) and high potassium and alkalinization as a positive control (activation), ionomycin (2 pM final) reference control and;

[0043] establishing a baseline for approximatelyu 30 seconds (often less than a minute) for each compound of unknown activity before a concentration of each compound is dispensed into cells (e.g. I OpL of drug to lOOpL of cells) and recorded for at least 5-10 minutes (typically 400 seconds; 6.8 minutes);

[0044] determining a maximum of calcium signal for each compound tested can be used to flag tested compounds which may be autofluorescent or interacting with calcium dye, wherein a compound which evidences a positive / favorable impact on calcium flow (increase in calcium influx) compared to the baseline is considered a potential / likely fertility agent. In preferred embodiments, the assay is performed in FLIPR.

[0045] In embodiments directed to a method of identifying a compound of unknown CatSper channel activity as an inhibitor of CatSper and consequently, a potential contraceptive (inhibitor of CatSper channel activity) agent, the method comprises the steps of plating a population of stable transfected cells according to the present invention and incubating these cells for about 18-30 hours, often 24 hours in cell media;

[0046] removing the cell media from the incubated cells and replacing the cell media with buffer solution (e.g. Hanks buffered saline solution HBSS);

[0047] adding calcium dye to the cells and incubating the cells in the buffered saline solution for approximately 15 minutes to one hour, often 30 minutes at approximately body temperature (about 37°C) and then for approximately 15 minutes to one hour, often 30 minutes at room temperature;

[0048] establishing a baseline for 30 seconds for each compound of unknown activity before a concentration of each compound is dispensed into cells (e.g. 5pL of drug to 50pL of cells) and recorded for at least 5-10 minutes (typically 400 seconds; 6.8 minutes) in Phase I reading;

[0049] recording for another 5-10 minuets (typically 400 seconds; 6.8 minutes) after adding high potassium and alkalinization as CatSper activating conditions in Phase 2 reading;

[0050] calculating activity cutoffs for each phase of the calcium response;

[0051] determining Max-Min in the phase 2 as inhibition of activation with those with 0.1% DMSO (vehicle) addition in Phase 1 with activator addition in Phase 2 as negative control (no inhibition) and vehicle in Phase 1 but with water addition (no activator) and lonomycin (2 uM final) in Phase 2 as positive control (full inhibition) and reference control, respectively;

[0052] determining max-min or max in Phase 1 reading to flag compounds that either increase or decreases calcium signal prior to the addition of activator. In preferred embodiments, the assay used is performed in FLIPR.

[0053] The above-described embodiments and other aspects of the invention may be readily gleaned from the description of the invention which follows.

[0054] Brief Description of the Figures

[0055] FIGURE 1 shows vector OsEukCatAl-hCatSper3 in phCMV3 (6161 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSper3 in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSper3 includes O. Sinensis EukCatl N-terminal soluble domain, human CatSper3 transmembrane domains 1-6, O. Sinensis EukCatl C-terminal soluble domain, and eGFP or mCherry marker. FIGURE 2 shows Novel classes of single-domain (ID) voltage-gated cation channels (EukCats) widely distributed in eukaryotic phytoplankton Taxa. FIGURE 2A shows the maximum likelihood (ML) phylogenetic tree ID voltage-gated cation channels including BacNavs, CatSpers, and EukCats. Three distinct classes (EukCatA-C) are observed. ML bootstrap values (>70) and Bayesian posterior probabilities (>0.95) are indicated on selected nodes. Branch colors denote taxonomic group for EukCat channels. FIGURE 2B shows a schematic diagram of a EukCat channel with selectivity filter of Phaeodactylum tricornutum protein (PtEUKCATAl; protein ID 43878) is also shown (lower left), indicating the position of transmembrane domains, selectivity filter, and coiled-coil domain (CC) (from Helliwell et al., 2019). Soluble cytoplasmic domains of two ID EukCatA channels from diatom, PtEUKCATAl and Odontella Sinensis OsEUKCATAl (protein ID CAMPEP O 183296650) are used to generate chimeric CatSper in this study.

[0056] FIGURE 3 shows an illustration of the domain structure and amino acid sequence of Os-hs chimeric CatSper3 protein. FIGURE 3A Shows the domain structure of chimeric human CatSper3 containing N-terminal soluble domain ofEukCatAl from 0. sinensis, six transmembrane (TM) domains of CatSper3 from H. sapiens, and a C-terminal soluble domain of EukCatAl from 0. sinensis. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx. FIGURE 3B shows the amino acid sequence of chimeric human CatSper3 containing N-terminal soluble domain of EukCatAl from O. sinensis (grey), six transmembrane (TM) domains of CatSper3 from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from 0. sinensis (grey). Sl-S6 regions are underlined (black) at the sequence while non-underlined sequences between S regions represent linking structures (fusion peptides). The sequence shown in FIGURE 3B is identical to SEQ ID NO:3 of TABLE 1.

[0057] FIGURE 4 shows the plasma membrane localization of Os-huCatSper3 channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red. FIGURE 5 shows that the recombinant expression of human CatSper channel in mammalian cells do not reach the cell surface. FIGURE 5A shows an illustration of domain structures and confocal images of full-length human CatSper. Topological illustration includes N- and C-terminal soluble domains and six transmembrane (TM) domains from H. sapiens. Shown are confocal images from full-length human Castperl, 2, 3 and 4 in COS7 cells (left to right). The results from HEK293 or 293T cells are in the same pattern. Subcellular localization of CatSper proteins (green) show a typical pattern of ER / Golgi but not a rim of the cells as shown in cells expressing OsEukCatAl (see FIGURE 6) or OsEukCatAl-hCatSper3 (i.e., Os-hCatSper3, see FIGURE 4). FIGURE 5B shows an illustration of domain structure and confocal images of human CatSper TM only proteins. Topological illustration shows truncated channel containing six TM domains from H. sapiens that lacks N-terminal and C-terminal soluble domains. Confocal images from human Castperl, 2, 3 and 4 TM only (left to right). Subcellular localization of TM-only CatSper proteins (green) show a typical pattern of ER / Golgi but not a rim of the cells as shown in cells expressing OsEukCatAl (see FIGURE 6) or OsEukCatAl -hCatSper3 (i.e., Os-hCatSper3, see FIGURE 4).

[0058] FIGURE 6 shows that recombinant expression of EukCatAl channel from 0. sinensis in mammalian cells are localized at the cell surface. Shown is a representative image of HEK293 cells expressing OsEukCatAl protein in fusion with eGFP shown in green (right pnel). A surface marker (Cholera Toxin B, CTxB) is shown in red (left panel).

[0059] FIGURE 7 shows that high K+(pH8.6) affects intracellular Ca2+signaling in HEK293 cells expressing Os-huCatSper3 (eGFP positive). FIGURE 7A shows representative measurements of cytosolic Ca signals, displayed as Fura2 ratio (F340 / F380), showing the effect of high K+(pH8.6) in HEK293 cells expressing Os-huCatSper3 (eGFP positive) for 80 secs. FIGURE 7B shows representative measurements of cytosolic Ca signals, displayed as Fura2 ratio (F340 / F380), showing non-effect of high K+(pH8.6) in non-transfected HEK293 cells. Ca ionophore ionomycin (10 M) was applied as a control stimulus.

[0060] [Ca2+]i

[0061] Ionomycin caused [Ca ]i rise in the presence of extracellular Ca by Ca influx.

[0062] FIGURE 8 shows a patch clamp recording HEK293 cells expressing Os-huCatSper3 detects ICatSper-like current not detected in non-transfected HEK293 cells. The traces show representative time courses of IcatSper measured in control solutions (BL, baseline), nominally divalent-free (DVF) solutions, and wash-off from DVF to BL at -100 mV. FIGURE 9 shows phCMV3 (4251 bp) backbone plasmid used in this study for modifications of nucleic acid constructs encoding a calcium ion channel. Compact mammalian cell vector for high-level expression of C-terminally HA-tagged proteins has been modified including chimeric CatSper or a few conventional 4D voltage-gated calcium channel (Cav) followed by Thrombin and eGFP or mCherry with a 6xHis tag. All sequences were in frame and followed by a final stop codon before HA.

[0063] FIGURE 10 shows vector OsEukCatAl-hCatSperl in phCMV3 (6179 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSperl in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSperl includes O. Sinensis EukCatl N-terminal soluble domain, human CatSperl transmembrane domains 1-6, O. Sinensis EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0064] FIGURE 11 shows vector OsEukCatAl-hCatSper2 in phCMV3 (6206 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSper2 in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSper2 includes O. Sinensis EukCatl N-terminal soluble domain, human CatSper2 transmembrane domains 1-6, O. Sinensis EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0065] FIGURE 12 shows vector OsEukCatAl-hCatSper4 in phCMV3 (6152 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSper4 in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSper4 includes O. Sinensis EukCatl N-terminal soluble domain, human CatSper4 transmembrane domains 1-6, O. Sinensis EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0066] FIGURE 13 shows vector PtEukCatAl-hCatSperl in phCMV3 (6422 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSperl in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSperl includes P. tricornutum EukCatl N-terminal soluble domain, human CatSperl transmembrane domains 1-6, P. tricornutum EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0067] FIGURE 14 shows vector PtEukCatAl-hCatSper2 in phCMV3 (6449 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSper2 in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSper2 includes P. tricornutum EukCatl N-terminal soluble domain, human CatSper2 transmembrane domains 1-6, P. tricornutum EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0068] FIGURE 15 shows vector PtEukCatAl-hCatSper3 in phCMV3 (6404 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSper3 in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSper3 includes P. tricornutum EukCatl N-terminal soluble domain, human CatSper3 transmembrane domains 1-6, P. tricornutum EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0069] FIGURE 16 shows vector PtEukCatAl-hCatSper4 in phCMV3 (6395 bp). This vector allows for the expression in mammalian cells of a channel protein, in this case the chimeric human CatSper4 in fusion with a detectable marker such as eGFP or mCherry. Chimeric human CatSper4 includes P. tricornutum EukCatl N-terminal soluble domain, human CatSper4 transmembrane domains 1-6, P. tricornutum EukCatl C-terminal soluble domain, and eGFP or mCherry marker.

[0070] FIGURE 17 shows an illustration of domain organization and amino acid sequence of Os-Human chimeric CatSperl. FIGURE 17A shows the domain organization of chimeric human CatSperl containing N-terminal soluble domain of EukCatAl from O. sinensis, six transmembrane (TM) domains of CatSperl from H. sapiens, and a C-terminal soluble domain of EukCatAl from O. sinensis. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx. FIGURE 17B shows the protein sequence of chimeric human CatSperl containing N-terminal soluble domain of EukCatAl from O. sinensis (grey), six transmembrane (TM) domains of CatSperl from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from O. sinensis (grey). S1-S6 regions are underlined (black) at the sequence while non-underlined sequences between S regions represent linking structures. The sequence shown in FIGURE 17B is identical to SEQ ID NO:1 of TABLE 1.

[0071] FIGURE 18 shows an illustration of the domain organization and amino acid sequence of Os-Human chimeric CatSper2. FIGURE 18A shows a topological illustration of chimeric human CatSper2 containing N-terminal soluble domain of EukCatAl from O. sinensis, six transmembrane (TM) domains of CatSper2 from H. sapiens, and a C-terminal soluble domain of EukCatAl from O. sinensis. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx. FIGURE 18B shows the protein sequence of chimeric human CatSper2 containing N-terminal soluble domain of EukCatAl from O. sinensis (grey), six transmembrane (TM) domains of CatSperl from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from O. sinensis (grey). S1-S6 regions are underlined (black) at the sequence while non-underlined sequences between S regions represent linking structures. The sequence shown in FIGURE 18B is identical to SEQ ID NO:2 of TABLE 1.

[0072] FIGURE 19 shows an illustration of domain organization and amino acid sequence of Os-Human chimeric CatSper4. FIGURE 19A shows a topological illustration of chimeric human CatSper4 containing N-terminal soluble domain of EukCatAl from O. sinensis, six transmembrane (TM) domains of CatSper4 from H. sapiens, and a C-terminal soluble domain of EukCatAl from O. sinensis. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx. FIGURE 19B shows the protein sequence of chimeric human CatSper4 containing N-terminal soluble domain of EukCatAl from O. sinensis (grey), six transmembrane (TM) domains of CatSper4 from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from O. sinensis (grey). S1-S6 regions are underlined (black) at the sequence while non-underlined sequences between S regions represent linking structures. The sequence shown in FIGURE 19B is identical to SEQ ID NO:4 of TABLE 1.

[0073] FIGURE 20 shows an illustration of domain organization and amino acid sequence of Pt-Human chimeric CatSperl. FIGURE 20A shows a topological illustration of chimeric human CatSperl containing N-terminal soluble domain of EukCatAl from / < tricornutum, six transmembrane (TM) domains of CatSperl from H. sapiens, and a C-terminal soluble domain of EukCatAl from P. tricornutum. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx.

[0074] FIGURE 20B shows the protein sequence of chimeric human CatSperl containing N-terminal soluble domain of EukCatAl from P. tricornutum (grey), six transmembrane (TM) domains of CatSperl from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from P. tricornutum (grey). S1-S6 regions are underlined (black) at the sequence while nonunderlined sequences between S regions represent linking structures. The sequence shown in FIGURE 20B is identical to SEQ ID NO:5 of TABLE 1.

[0075] FIGURE 21 shows an illustration of domain organization and amino acid sequence of Pt-Human chimeric CatSper2. FIGURE 21A shows a topological illustration of chimeric human CatSper2 containing N-terminal soluble domain of EukCatAl from P. tricornutum, six transmembrane (TM) domains of CatSperl from H. sapiens, and a C-terminal soluble domain of EukCatAl from P. tricornutum. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a 2+ conserved homologous amino acid sequence, which selectively permits Ca influx.

[0076] FIGURE 21B shows the protein sequence of chimeric human CatSper2 containing N-terminal soluble domain of EukCatAl from P. tricornutum (grey), six transmembrane (TM) domains of CatSper2 from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from P. tricornutum (grey). S1-S6 regions are underlined (black) at the sequence while nonunderlined sequences between S regions represent linking structures. The sequence shown in FIGURE 21B is identical to SEQ ID NO:6 of TABLE 1.

[0077] FIGURE 22 shows an illustration of domain organization and amino acid sequence of Pt-Human chimeric CatSper3. FIGURE 22A shows a topological illustration of chimeric human CatSper3 containing N-terminal soluble domain of EukCatAl from P. tricornutum, six transmembrane (TM) domains of CatSper3 from H. sapiens, and a C-terminal soluble domain of EukCatAl from P. tricornutum. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx.

[0078] FIGURE 22B shows a protein sequence of chimeric human CatSper3 containing N-terminal soluble domain of EukCatAl from P. tricornutum (grey), six transmembrane (TM) domains of CatSper3 from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from P. tricornutum (grey). S1-S6 regions are underlined (black) at the sequence while nonunderlined sequences between S regions represent linking structures. The sequence shown in FIGURE 22B is identical to SEQ ID NO:7 of TABLE 1.

[0079] FIGURE 23 shows an illustration of domain organization and amino acid sequence of Pt-Human chimeric CatSper4. FIGURE 23A shows a topological illustration of chimeric human CatSper4 containing N-terminal soluble domain of EukCatAl from P. tricornutum, six transmembrane (TM) domains of CatSperl from H. sapiens, and a C-terminal soluble domain of EukCatAl from P. tricornutum. CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx.

[0080] FIGURE 23B shows a protein sequence of chimeric human CatSper4 containing N-terminal soluble domain of EukCatAl from P. tricornutum (grey), six transmembrane (TM) domains of CatSper4 from H. sapiens (black), and a C-terminal soluble domain of EukCatAl from P. tricornutum (grey). S1-S6 regions are underlined (black) at the sequence while nonunderlined sequences between S regions represent linking structures. The sequence shown in FIGURE 23B is identical to SEQ ID NO:8 of TABLE 1.

[0081] FIGURE 24 shows that recombinant expression of Os-huCatSperl channel in mammalian cells are localized at the cell surface. Shown is a representative image of HEK293 cells expressing OsEukCatAl protein in fusion with eGFP shown in green. A surface marker (Cholera Toxin B, CTxB) is shown in red.

[0082] FIGURE 25 shows the plasma membrane localization of Os-huCatSper2 channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red.

[0083] FIGURE 26 shows the plasma membrane localization of Os-huCatSper4 channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red.

[0084] FIGURE 27 shows the plasma membrane localization of Pt-huCatSperl channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red.

[0085] FIGURE 28 shows the plasma membrane localization of Pt-huCatSper2 channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red.

[0086] FIGURE 29 shows the plasma membrane localization of Pt-huCatSper3 channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red. FIGURE 30 shows the plasma membrane localization of Pt-huCatSper4 channel protein in HEK293 cells. The chimeric proteins detected by eGFP (green) are mostly co-localized with the surface marker (Cholera Toxin B, CTxB) shown in red.

[0087] FIGURE 31, similar to FIGURE 12 shows vector OsEukCatAl-hCATSPER4 in phCMV3 (6135 bp). This vector allows for the expression of a channel protein in mammalian cells, in this case the chimeric human hCATSPER4 in frame with a detectable marker mCherry.

[0088] Chimeric human hCATSPER4 includes the transmembrane domains of CATSPER4 (S1-S6) fused with N- and C-terminal cytosolic domains of O. sinensis EukCatl. A monoclonal stable cell line (El IL) is established using the construct.

[0089] FIGURE 32 shows that alkaline depolarization increases intracellular Ca signaling in a monoclonal cell line stably expressing Os-hCATSPER4 chimeric channel. FIGURE 32A shows representative measurements of cytosolic Ca signals by Fluo-4 as relative fluorescent unit, showing the effect of high K+and intracellular alkalinization on the channel by 135 mM KCl and 20 mM NH4C1. Ca ionophore ionomycin (10 pM) was applied as a control

[0090] 2+ stimulus. FIGURE 32B El IL clone shows the most robust and reproducible Ca response (Z’=0.54) to the alkaline depolarization activation from the baseline (RFUK8.6-RFUBL), establishing it as the homotetrameric hCATSPER4 cell line. Non-transfected, parental HEK293 cells do not show significant Ca response.

[0091] FIGURE 33 shows a patch clamp recording of magnesium blocking of IcatSpe ^ current in El IL cells stably expressing Os-hCATSPER4 chimeric channels. The IV curve shows the inward IcatSper measured when bath solution is switched from control bath solution containing 2+ 2+

[0092] Ca and Mg (baseline) to divalent-free (DVF) solutions, which is blocked by baseline solution with Mg only but nominal Ca free, and wash-off to baseline. Both bath and pipette solutions were kept at pH 7.3. The recording was held at 0 mM and measured in ramp from -120 mM to + 120 mV over 400 msec.

[0093] FIGURE 34 shows a patch clamp recording of alkaline activation of IcatSper-^Q current in El IL cells stably expressing Os-hCATSPER4 chimeric channels. The IV curve shows the inward IcatSper measured when bath solution is switched to divalent-free (DVF) solutions from 2+ 2+

[0094] control bath solution containing Ca and Mg (baseline), followed by increase in IcatSper in 2+

[0095] DVF with 20 mM NH4C1, which is blocked by baseline solution with Mg only but nominal 2+

[0096] Ca free, and wash-off to baseline. The pipette solution was kept at pH 6.0 while the bath solution was pH 7.3. The recording was held at 0 mM and measured in ramp from -120 mM to + 120 mV over 400 msec.

[0097] FIGURE 35, similar to FIGURE 10 shows vector OsEukCatAl-hCATSPERl in phCMV3 (6162 bp). This vector allows for the expression of a channel protein in mammalian cells, in this case the chimeric human hCATSPERl in frame with a detectable marker mCherry at the C-terminus. Chimeric human hCATSPERl includes the transmembrane domains (S1-S6) of hCATSPERl fused with N- and C-terminal cytosolic domains of O. sinensis EukCatl. A monoclonal stable cell line (E9H) is established using the construct.

[0098] FIGURE 36, similar to FIGURE 11, shows vector OsEukCatAl-hCATSPER2 in phCMV3 (6189 bp). This vector allows for the expression of a channel protein in mammalian cells, in this case the chimeric human hCATSPER2 in frame with a detectable marker mCherry at the C-terminus. Chimeric human hCATSPER2 includes the transmembrane domains (S1-S6) of hCATSPER2 fused with N- and C-terminal cytosolic domains of 0. sinensis EukCatl. A monoclonal stable cell line (F2L) is established using the construct.

[0099] FIGURE 37, similar to FIGURE 1, shows vector OsEukCatAl-hCATSPER3 in phCMV3 (6144 bp). This vector allows for the expression of a channel protein in mammalian cells, in this case the chimeric human hCATSPER3 in frame with a detectable marker mCherry at the C-terminus. Chimeric human hCATSPER3 includes the transmembrane domains (S1-S6) of hCATSPER3 fused with N- and C-terminal cytosolic domains of 0. sinensis EukCatl. A monoclonal stable cell line (D3H) is established using the construct.

[0100] FIGURE 38 shows that alkaline depolarization increases intracellular Ca in the monoclonal cell lines stably expressing Os-hCATSPERl, Os-hCATSPER2, or Os-hCATSPER3 chimeric channel. Representative measurements of cytosolic Ca signals by Fluo-4 as relative fluorescent unit, showing the effect of high K+and intracellular alkalinization on the channel by adding 135 mM KC1 and 20 mM NH4CI. Ca ionophore ionomycin (10 pM) was applied 2+ as a control stimulus. E9H, F8L, and D3H clones show the most robust and reproducible Ca responses from Os-hCATSPERl, Os-hCATSPER2, and Os-hCATSPER3 clones to the alkaline depolarization activation from the baseline (RFUKS.6-RFUBL) with Z’=0.51, 0.6, 0.59, respectively. FIGURE 39 shows vector hCav3.2-hCATSPERl-4-3-2 in phCMV3 (11,464 bp). This mammalian expression vector allows for the expression of the inline chimeric CatSper channel protein in the counterclockwise order under a CMV promoter, replacing each of the 4D human Cav3.2 with 6-TM of hCATSPERl, hCATSPER4, hCATSPER3, and hCATSPER2 in frame with mCherry marker. Chimeric hCATSPERl -4-3 -2 includes N- and C-terminal cytosolic domains and the loops between each 6TM domains of human Cav3.2 (see also FIGURE 41 A).

[0101] FIGURE 40 shows vector hCav3.2-hCATSPERl-4-3-2 in pCW57.1 (15,112 bp). This Lenti viral vector allows for the transduction and expression of the inducible inline chimeric CatSper channel protein in the counterclockwise order under the control of tetracycline, replacing each of the 4D human Cav3.2 with 6-TM of hCATSPERl, hCATSPER4, hCATSPER3, and hCATSPER2 in frame with mCherry marker. Chimeric hCATSPERl -4-3-2 includes N- and C-terminal cytosolic domains and the loops between each 6TM domains of human Cav3.2 (see also FIGURE 41 A).

[0102] FIGURE 41 shows an illustration of domain organization and amino acid sequence of hCav3.2-hCATSPERl-4-3-2 chimera. FIGURE 41A shows a topological illustration of chimeric hCav3.2-hCATSPERl-4-3-2 containing includes N- and C-terminal cytosolic domains and the loops between each 6TM domains of human Cav3.2. Each CatSper has two physiologically distinctive regions, a voltage-sensing domain (S1-S4) and a pore-forming region (S5-S6). S4 contains several (two to six) positively charged amino acid residues that serve as voltage sensors. Voltage slopes move S4, resulting in conformational changes that open and close the channel pore. S5 and S6 are linked by a short and hydrophobic cyclic structure that contains a conserved homologous amino acid sequence, which selectively permits Ca influx. FIGURE 41B shows the protein sequence of chimeric hCav3.2-hCATSPERl-4-3-2 channel, six transmembrane (TM) domains of each CATSPER poreforming subunits from H. sapiens (black) are highlighted in beige (CATSPER1), light green (CATSPER4), pink (CATSPER3), and red (CATSPER2) and N-and C-terminal cytosolic domains and the loops between the four 6TM domains are from human Cav3.2 (blue). The sequence shown in FIGURE 41B is identical to SEQ ID NOV of TABLE 1. FIGURE 42 shows that recombinant hCav3.2-hCATSPERl-4-3-2 chimeric channels in mammalian cells are localized at the cell surface. Shown on the left are representative images of the monoclonal cell line expressing inducible hCav3.2-hCATSPERl-4-3-2 chimeric channels before and after 48 h after doxycycline induction. The chimera was detected by anti-mCherry with nucleus in blue. Shown on the right is a representative Western blot detection of hCav3.2-hCATSPERl-4-3-2-mCherry protein from the monoclonal cell line after doxycycline induction.

[0103] 2+

[0104] FIGURE 43 shows that alkaline depolarization increases intracellular Ca signaling in the monoclonal cell line which express hCav3.2-hCATSPERl-4-3-2 chimeric channel. FIGURE 43A shows representative measurements of cytosolic Ca signals by Fluo-4 as relative fluorescent unit, showing the effect of high K+and intracellular alkalinization on the channel by 135 mM KC1 and 20 mM NH4CI mainly on the cells induced by doxycycline treatment.

[0105] FIGURE 43B shows that non-transfected, parental HEK293 cells do not show significant Ca response. Ca ionophore ionomycin (10 pM) was applied as a control stimulus.

[0106] FIGURE 44 shows a patch clamp recording of IcatSper-^Q current in the monoclonal HEK293 cells expressing hCav3.2-hCATSPERl-4-3-2 chimeric channel. The current elicited under the ramp protocol shows the inward IcatSper measured when bath solution is switched to divalent-free (DVF) solutions from control bath solution containing Ca and Mg (baseline), which is not activated by progesterone (P4, 500 nM) but partially blocked by nonspecific CatSper inhibitor NNC 55-0396 (2 pM) and almost completely blocked by the baseline solution with 1 mM Mg only but nominal Ca free, and wash-off to baseline. Both the bath and the pipette solutions were kept at pH 7.3. The recording was held at 0 mM and measured in ramp from -120 mM to + 120 mV over 400 msec.

[0107] FIGURE 45 shows a FLIPR calcium assay for activator screen with the monoclonal HEK293 cells expressing Os-hCATSPER4 chimeric channel. The calcium trace under the single-phase activation protocol shows an example of the increase of intracellular calcium ([Ca ]i) concentration by a hit molecule (X, blue) compared to the known activating condition (alkaline depolarization by KC1 and NH4C1, red) as a positive control and vehicle (0.1% DMSO, green) as a negative control. FIGURE 46 shows a FLIPR calcium assay for inhibitor screen with the monoclonal HEK293 cells expressing Ox-hCATSPER4 chimeric channel. The calcium trace under the two-phase inhibitor protocol shows an example of the decrease of [Ca ]i by a hit molecule (Y) in triplicates in response to the activating condition (alkaline depolarization by KC1 and NH4C1) compared to vehicle-treated condition.

[0108] Detailed Description of the Invention

[0109] In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techiques are explained fully in the literature. See, e.g., Sambrook et al, 2001, " Molecular Cloning: A Laboratory Manual"; Ausubel, ed., 1994, " Current Protocols in Molecular Biology" Volumes I-III; Celis, ed., 1994, " Cell Biology: A Laboratory Handbook" Volumes I-III; Coligan, ed., 1994, " Current Protocols in Immunology" Volumes I-III; Gait ed., 1984, " Oligonucleotide Synthesis"; Hames & Higgins eds., 1985, " Nucleic Acid Hybridization"; Hames & Higgins, eds., 1984, " Transcription And Translation"; Freshney, ed., 1986, " Animal Cell Culture"; IRL Press, 1986, " Immobilized Cells And Enzymes"; Perbal, 1984, " A Practical Guide To Molecular Cloning."

[0110] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0111] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It must be noted that as used herein and in the appended claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0112] Furthermore, the following terms shall have the definitions set out below.

[0113] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and singlestranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, such as coding regions, and non-coding regions such as regulatory sequences (e.g., promoters or transcriptional terminators). A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a plasmid vector, such as an expression or cloning vector, or a fragment.

[0114] Restriction endonucleases are enzymes that cleave DNA at well-defined sequences. They are used in recombinant DNA technology, for example, to generate specific DNA fragments that are readily joined through the action of DNA ligase to other DNA. They are used in recombinant DNA technology, for example, to generate specific DNA fragments that are readily joined through the action of DNA ligase to other DNA fragments generated by digestion with the same restriction endonuclase.

[0115] As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" also includes molecules which contain more than one polypeptide joined together such as chimeric peptide CatSper channels as described herein. These polypeptides may be joined by a small oligopeptide linker group, by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The amino acid residues described herein are preferred to be in the " L" isomeric form. However, in certain instances, residues in the " D" isomeric form may be substituted for any L-amino acid residue, as long as the desired function is retained by the polypeptide. NH2refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.

[0116] The term “coding sequence” is defined herein as a portion of a nucleic acid sequence that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by a ribosome binding (or Shine-Dalgamo) site and a translation initiation codon (usually AUG) in prokaryotes, or by the AUG start codon in eukaryotes located at the start of the open reading frame, usually near the 5’ - end of the mRNA, and a translation terminator sequence (one of the nonsense codons: UAG, UGA, or UAA) located at and specifying the end of the open reading frame, usually near the 3’ - end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0117] A “stop codon” or “termination codon” is a nucleotide triplet within messenger RNA that signals a termination of translation. Proetins are unique sequences of amino acids, and most codons in messenger RNA correspond to the addition of an amino acid to a growing protein chain — stop codons signal the termination of this process, releasing the amino acid chain. In the standard genetic code, there are three stop codons: UAG (in RNA) / TAG (in DNA) (also known as an "amber" stop codon), UAA / TAA (also known as an "ochre" stop codon), and UGA / TGA (also known as an "opal" or "umber" stop codon). Several to this predominant group are known. The use of a stop codon in the present invention will normally stop or terminate protein synthesis. However, there are mutations in tRNAs which allow them to recognize the stop codons, causing ribosomes to read through the stop codon, allowing synthesis of peptides encoded downstream of the stop codon.

[0118] A "heterologous" region is an identifiable segment of nucleic acid within a larger nucleic acid molecule that is not found in association with the larger molecule in nature. A “heterologous” peptide is a peptide which is an identifiable segment of a polypeptide that is not found in association with the larger polypeptide in nature. An "origin of replication", used within context, normally refers to those DNA sequences that participate in DNA synthesis by specifying a DNA replication initiation region. In the presence of needed factors (DNA polymerases, and the like) an origin of replication causes DNA associated with it to be replicated. An appropriate replication origin endows many commonly used plasmid cloning vectors with the capacity to replicate independently of the bacterial chromosome.

[0119] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. The promoter sequence includes the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found DNA sequences responsible for the binding of RNA polymerase and any of the associated factors necessary for transcription initiation. In bacteria, promoters normally consist of -35 and -10 consensus sequences and a more or less specific transcription initiation site. Eukaryotic promoters will often, but not always, contain " TATA" boxes and " CAT" boxes. Bacterial expression vectors (usually plasmids or phages) typically utilize promoters derived from natural sources, including those derived from the E. coli Lactose, Arabinose, Tryptophan, and ProB operons, as well as others from bacteriophage sources. In viruses, promoters are DNA sequences that functions as switches for gene activation. These promoters are binding sites for DNA polymerase and other proteins which are necessary for transcription.

[0120] In bacteria, transcription normally terminates at specific transcription termination sequences, which typically are categorized as rho-dependent and rho-independent (or intrinsic) terminators, depending on whether they require the action of the bacterial rho-factor for their activity. These terminators specify the sites at which RNA polymerase is caused to stop its transcription activity, and thus they largely define the 3 ’-ends of the RNAs, although sometimes subsequent action of ribonucleases further trims the RNA. Termination of transcription in viral vectors involves terminator sequences that signal the end of RNA synthesis. These sequences are often derived from viral genomes.

[0121] An “antibiotic resistance gene” refers to a gene that encodes a protein that renders a bacterium resistant to a given antibiotic. In the present invention, antibiotic resistance genes may be used to ensure the maintenance within bacteria of plasmids which encode for chimeric CatSper channel proteins.

[0122] “Reverse transcription and PCR” are presented as a means of amplifying the nucleic acid sequences encoding chimeric peptides described herein. “Reverse transcription” refers to the process by which a DNA copy of an RNA molecule (or cDNA) is produced by the action of the enzyme reverse transcriptase. In the present application, reverse transcription is used to produce a DNA copy of RNA sequences of chimeric peptides. The reverse transcriptase enzyme requires a primer be annealed to the RNA.

[0123] The term “PCR” refers to the polymerase chain reaction, a technique used for the amplification of specific DNA sequences. The term “PCR primer” refers to DNA sequences (usually synthetic oligonucleotides) able to anneal to a target DNA, thus allowing a DNA polymerase (e.g. Taq DNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are used in the polymerase chain reaction to initiate DNA synthesis on each of the two strands of a DNA and to thus amplify the DNA segment between the two primers.

[0124] A cell has been "transformed" by exogenous or heterologous DNA when such DNA, especially including an expression vector which expresses a chemical CatSper channel as described herein has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell, but preferably forms a stably transformed cell line. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid, which normally replicate independently of the bacterial chromosome by virtue of the presence on the plasmid of a replication origin. With respect to eukaryotic cells, a “stably transformed cell” is one in which the transforming DNA such as an expression vector has become integrated into the foreign genetic material of the transformed cell (i.e., into the genome of the foreign cells) thus allowing the cells to express the new gene over many generations. Thus, in stably transfected cell, a chromosome is transfected so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA, in the present application the chimeric CatSper channels of the present invention. It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding the polypeptide(s) of the present invention, which code for a polypeptide having the same amino acid sequence as the sequences disclosed herein, but which degenerate to the nucleic acids disclosed herein. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.

[0125] A nucleic acid molecule is "operatively linked" to, or “operably associated with” an expression control sequence when the expression control sequence controls and regulates the transcription and translation of a nucleic acid sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the nucleic acid sequence to be expressed and maintaining the correct reading frame to permit expression of the nucleic acid sequence under the control of the expression control sequence and production of the desired product encoded by the nucleic acid sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

[0126] The present invention is further described with respect to experiments described in the following examples.

[0127] EXAMPLES

[0128] Example 1

[0129] Materials and Methods

[0130] Molecular Cloning

[0131] Bacterial strains

[0132] NEB® 10-P (NEB) bacterial strains were used for molecular cloning.

[0133] Mammalian cell expression constructs

[0134] Cloned DNAs encoding Phaeoodactylum tricornutum EUKCATA1 (PtEUKCATAl, protein ID 43878) or Odontella sinensis EUKCATA1 (OsEUKCATAl, protein ID

[0135] CAMPEP O 183296650) in frame with EGFP have been expressed in HEK293 cells and produced functional Na and Ca selective channels (Helliwell, Chrachri et al. 2019). DNAs encoding full length or the transmembrane human CatSperl, 2, 3, or 4 were cloned into phCMV3 to express C-terminal HA-tagged proteins (phCMV3-huCatSperl, 2, 3, or 4-FL or -TM) by the Chung Laboratory at Yale University School of Medicine. EukCatA-CatSper chimeric constructs in phCMV3 vector were generated using NEBuilder® HiFi DNA Assembly Kit (NEB) as following (see also FIGURE 9). First, PtEUKCATAl or OsEUKCATAl was subcloned into phCMV3 in frame with a DNA fragment encoding Thrombin cleavage site followed by EGFP (or mCherry), 6 repeats of HIS tag (6XHIS) and a stop codon at the upstream of HA sequences in phCMV3. Second, the transmembrane domain of PtEUKCATAl or OsEUKCATAl was replaced with a DNA fragment encoding the transmembrane domains of either human CatSperl, 2, 3, or 4 to finally yield phCMV3-OsCatSperl, 2, 3 or 4 and phCMV3-PtCatSperl, 2, 3 or 4 in fusion with either EGFP or mCherry (see also FIGURE 1 and FIGURES 9- 15). Amino acid sequences of chimeric EukCatA-CatSper proteins expressed by these chimeric constructs were shown in FIGURE 3 and FIGURES 17-23

[0136] Cell culture

[0137] Mammalian cell lines

[0138] HEK293 cells (ATCC; derived from female embryonic kidney) or COS7 cells (ATCC; derived from African green monkey kidney) were cultured in DMEM (GIBCO) containing 10% FBS (Thermo fisher) and lx Pen / Strep (GIBCO) at 37 °C, 5% CO2 condition.

[0139] Transient transfection

[0140] HEK293 cells and COS7 cells were transiently transfected with plasmids phCMV3-Pt / OsCatSperl, 2, 3 or 4 or phCMV3-huCatSperl, 2, 3, or 4-FL or -TM, respectively.

[0141] Lipofectamine 2000 (Invitrogen) was used for transfection following the manufacturer’s instruction. Transfected cells were used for immunocytochemistry.

[0142] Immunocytochemistry

[0143] Alexa Fluro 555 Conjugated Cholera Toxin Subunits B (CTxB, Invitrogen C34776) was used to label HEK293 plasma membrane (i.e. surface marker). 24 h after transfection, HEK293 cells were washed twice with pre-warmed PBS, then labeled with 2.5ug / ml CTxB containing HBSS at 37 °C for 15 min. Labelled cells were further washed with PBS and fixed with 4% formaldehyde for 15 min at room temperature. Coverslips were mounted with Prolong Gold Antifade Mount (ThermoFisher) and imaged with LSM710 using Plan-Apochrombat 63X / 1.40 objective lens (Carl Zeiss). COS7 cells were transfect with an indicated plasmids for 24 h and fixed with 4% formaldehyde for 15 min at room temperature. Fixed samples were permeabilized using 0.1% Triton X-100 in PBS at RT for 10 minutes, washed in PBS, and blocked with 3% BSA in PBS at RT for 1 h. Cells were stained with anti-HA rabbit primary antibody (CST 3724, 1:500) at 4 °C overnight. After washing in PBS, the samples were incubated with goat anti-rabbit Alexa488 (Invitrogen, 1: 1,000) in 3% BSA in PBS at RT for 1 hr. Immunostained samples were mounted with Prolong gold (Invitrogen), followed by imaging with LSM710 using Plan-Apochrombat 63X / 1 (Carl Zeiss). Represented images of each chimeric protein expressed in HEK293 cells in FIGURE 4A and FIGURES 24-29 demonstrates the plasma membrane localization of each recombinant protein.

[0144] Ca2+imaging

[0145] HEK293 cells expressing each chimeric channel in fusion with EGFP or mCherry were loaded with calcium indicators (Fura-2 AM or Fluo-4 AM, Life Technologies) at 2 or 2.5 mM and 0.02% pluronic acid-127 (F127, Life Technologies) for 30 min at 37°C. After loading, cells were washed twice and then incubated for a further 30 minutes with DMEM at 37°C. For ratiometric imaging of Fura-2, the excitation light was filtered through an Ultra High Speed Wavelength Switcher (Lambda DG-4 Plus; Sutter instruments, Novato, CA) to provide wavelengths of 340 and 380 nm. Emission light from individual cells was passed through an emission filter of 510 nm, and captured by a digital camera (pco.edge sCMOS) equipped in Axio observer Z1 microscope (Carl Zeiss). For a single excitation wavelength of Fluo-4, the excitation light was filtered through a 460-490 nm band pass filter and the emission light passed through a 510-550 nm band pass filter and captured by a high-speed digital camera (pco-edge sCMOS) equipped in Axio observer Z1 microscope (Carl Zeiss). DMEM was removed by perfusion with high pH depolarization using pH8.6 135mM

[0146] K+buffer (135 mM KC1, 5 mM NaCl, 1 mM MgSO4, 2 mM CaCl2, 20 mM HEPES, 5 mM Glucose, 10 mM lactic acid, 1 mM Na pyruvate, pH8.6). A representative trace from HEK293 cells expressing Os-huCatSper3 is shown in FIGURE 7A. For HEK293 cells not expressing the chimeric proteins, 10 mM ionomycin was added at the end of imaging as shown in FIGURE 7B.

[0147] Electrophysiological recording of HEK293 expressing chimeric human CatSper The chimeric CatSper channels expressed in HEK293 cells were recorded with the Port-a-Patch (Nanion) in whole cell configuration using 3-5 MQ chip (see also FIGURE 8). The standard bath solution was (mM): 140 NaCl, 4 KC1, 2 CaC12, 1 MgC12, 5 D-Glucose monohydrate, 10 HEPES, pH 7.4 with NaOH. After break-in, the access resistance was 12-24 MQ. The standard pipette solution was (mM): 10 NaCl, 110 CsF, 10 EGTA, 10 HEPES, pH 7.2 with CsOH. ICatSper was measured as monovalent current in divalent-free conditions (mM): 140 NaCl, 4 KC1, 5 D-Glucose monohydrate, 10 HEPES, pH 7.4 with NaOH.

[0148] Results / Conclusions Drawn From Example 1

[0149] Full-length or the transmembrane domains of human CatSperl-4 are retained intracellularly

[0150] Previous attempts to express each pore-forming (a) CatSper (1-4) subunit in full length in heterologous systems failed to target the protein(s) on the plasma membrane, resulting in their confinement to the ER and Golgi (Chung, Navarro et al. 2011, Chung, Miki et al. 2017) (see also FIGURE 5A). We generated truncated CatSper (1-4) without both N- and C-terminal soluble domains (i.e. TM-only channel) and found similar results (FIGURE 5B), suggesting that the intracellular domains do not particularly contain ER / Golgi retention signals.

[0151] Interspecies chimeric channel that contains human CatSper TM domains traffic to the surface

[0152] Recently, Brownlee Laboratory at Marine Biological Association, UK, reported novel eukaryotic ID voltage-gated non-selective cation channels distantly related to NaChBac and CatSper from multiple marine phytoplankton and named them as EukCat channels (Helliwell, Chrachri et al. 2019). Intrigued by their findings that two of these diatom ID channels from EukCatA class - Phaeoodactylum tricornutum EUKCAT Al (PtEUKCATAl, protein ID 43878) or Odontella sinensis EUKCATA1 (OsEUKCATAl, protein ID

[0153] CAMPEP O 183296650) - can be functionally expressed in HEK293 with detectable currents by electrophysiological recordings (FIGURE 2), we hypothesized the soluble cytoplasmic domains of these ID EukCats contain intrinsic signal motif(s) to allow tetrameric channel assembly and traffic to the plasma membrane. To test this idea, we first confirmed the plasma membrane localization of OsEUKCATAl in HEK293 cells (FIGURE 6 and unpublished data). Then, we generated DNA constructs that encode interspecies chimeric channels wherein the transmembrane and pore domains of each human CatSper is fused to soluble domains of either OsEukCatAl or PtEukCatAl in fusion with a detectable marker such as eGFP or mCherry (FIGURES 1, 3 and 10-23). All these eGFP chimeric channels successfully traffic to the plasma membrane of HEK293 cells, which are co-localized with Alex555-labeled Choleratoxin B (CTxB) (FIGURES 4 and 24-30). These results are in stark contrast to the full-length or TM-only recombinant CatSper 1-4, which show the typical ER / Golgi localizations (FIGURE 5).

[0154] The chimeric CatSper channel in HEK293 cells form functional calcium channel To test whether the chimeric CatSper channels localized in the plasma membrane in HEK293 cells form function channel that flux calcium ions, we performed Ca imaging with Fura-2, a calcium indicator that enables to detect the changes in the intracellular calcium levels ratiometrically. It was previously reported that CatSper channels in both mouse and human sperm cells are activated by depolarization and intracellular alkalinization (Kirichok, Navarro et al. 2006, Lishko, Botchkina et al. 2011, Strunker, Goodwin et al. 2011). Therefore, we stimulated the chimeric channel by applying high potassium and high pH media to the cells. HEK293 cells transiently expressing Pt- or Os-huCatSper3 in fusion with eGFP was Fura-2 loaded. eGFP-positive cells show an increase of intracellular calcium levels indicated by an increase of F340 / F380 signal for about 60 s upon high K+and high pH stimuli (FIGURE 7A). By contrast, eGFP-negative cells did not respond to the same conditions but showed a dramatic increase of F340 / F380 signal when a calcium ionophore, ionomycin, is added to the cells (FIGURE 7B). Furthermore, patch-clamp recording of HEK293 cells expressing the chimeric channel (Pt-huCatSper3) detected IcatSper-^-Q current carried by Na+ ion in DVF as is typical for Ca 'selective channels (FIGURE 8). All these results demonstrate the proof-of-principle and feasibility of reconstitution of human CatSper channel in chimeric forms in mammalian cells suitable for direct and high throughput screening of small molecule modulators.

[0155] Discussion

[0156] To our best knowledge, this study is the first to report functionally expressed CatSper channel from any species in mammalian cells. These chimeric channels supposedly form tetrameric channels (i.e. homotetramer) as do most ID voltage-gated channels such as voltage-gated potassium channels (Kv). In 4D voltage-gated sodium or calcium channels (Navor Cav), there are four repeats of 6 TM domains, thus taking the form of tetramer as well. Thus, these chimeric channels will be not only useful to launch the first series of direct, high throughput screenings of human CatSper for the purpose of developing a male contraceptive and / or a fertility enhancer. Simultaneously, the current chimeric forms will be particularly useful in studying different portions or domains of the CatSper channel proteins, for example, to elucidate the mechanisms underlying ion-selectivity or ligand-selectivity or gating mechanisms conferred by a portion, or a domain of a specific subunit. Moreover, these chimeric channels can be used to validate low affinity modulators of CatSper channel previously identified by FLIPR screening of human sperm (Carlson, Burnett et al. 2009, Gruber, Johnston et al. 2020, Carlson, Georg et al. 2021) or by electrophysiology studies (Miller, Mannowetz et al. 2016, Mannowetz, Miller et al. 2017, Jeschke, Biagioni et al. 2021, Rahban, Rehfeld et al. 2021). Ultimately, the information generated from these individual chimeric CatSperl, 2, 3, or 4 homotetrameric channels will lead to generation of a structure- assisted heterotetrametric CatSper that would better mimic the pore of the native CatSper but in minimal format for further screening and validation of identified molecules.

[0157] TABLE 1 Amino acid sequences of chimeric CatSper channels from Example 1 Chimeric channel Amino acid sequence

[0158] Os-huCatSperl MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID NO:1 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLLAFETFIF FWCLNTVMLVAQTFAEVEIRGEWYFMALDSIFFCIYWEALLKIIALG LSYFFDFWNNLDFFIMAMAVLDFLLMQTHSFAIYHQSLFRILKVFKSLR ALRAIRVLRRLSFLTSVQEVTGTLGQSLPSIAAILILMFTCLFLFSAVL RALFRKSDPKRFQNIFTTIFTLFTLLTLDDWSLIYMDSRAQGAWYIIPI LVIYIIIQYFIFLNLVITVLVDSFQALHEDERAKLHGTYESESEDIEDD EPDSEEQIKALETQIEELTRIQESTMMTLEYLTQQLQMVHLQQNQTDS

[0159] Os-huCatSper2 MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQID NO:2 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLPLFKNFII FLVFLNTIILMVEIELLESTNTKLWPLKLTLEVAAWFILLIFILEILLK WLSNFSVFWKSAWNVFDFWTMLSLLPEVWLVGVTGQSVWLQLLRICR VLRSLKLLAQFRQIQIIILVLVRALKSMTFLLMLLLIFFYIFAVTGVYV FSEYTRSPRQDLEYHVFFSDLPNSLVTVFILFTLDHWYALLQDVWKVPE VSRI FSS I YFILWLLLGS I I FRS I IVAMMVTNFQALHEDERAKLHGTYE SESEDIEDDEPDSEEQIKALETQIEELTRIQESTMMTLEYLTQQLQMVH LQQNQTDS

[0160] Os-huCatSper3 MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID NO:3 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLRFFKI IMI STVTSNAFFMALWTSYDIRYRLFRLLEFSEIFFVSICTSELSMKVYVDP INYWKNGYNLLDVI I I IVMFLPYALRQLMGKQFTYLYIADGMQSLRILK LIGYSQGIRTLITAVGQTVYTVASVLLLLFLLMYIFAILGFCLFGSPDN GDHDNWGNLAAAFFTLFSLATVDGWTDLQKQLDNREFALSRAFTIIFIL LASFIFLNMFVGVMIMHTEALHEDERAKLHGTYESESEDIEDDEPDSEE QIKALETQIEELTRIQESTMMTLEYLTQQLQMVHLQQNQTDS

[0161] Os-huCatSper4 MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID NO:4 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLPAFQLLLA LLLVINAITIALRTNSYLDQKHYELFSTIDDIVLTILLCEVLLGWLNGF WIFWKDGWNILNFIIVFILLLRFFINEINIPSINYTLRALRLVHVCMAV EPLARI IRVILQSVPDMANIMVLILFFMLVFSVFGVTLFGAFVPKHFQN IQVALYTLFICITQDGWVDIYSDFQTEKREYAMEIGGAIYFTIFITIGA FIGINLFVIWTTNLEALHEDERAKLHGTYESESEDIEDDEPDSEEQIK

[0162]

[0163] ALETQIEELTRIQESTMMTLEYLTQQLQMVHLQQNQTDS Pt-huCatSperl MATMQGIVENGQDKWEPEKLEDANRNQPGKRVRIMISSSLLQEKLQTP SEQ ID 1X10:5 ERLETVHRRDESLQGGDEEYSSHTAATTSADASSSNLSVRSVHHDEAED PSDSS FSAI PEDTAPACNE KVITSSIKSHASNERSNSEERKKEKPPTLP PSYASTIRKTRQFAGKLVNNLAFETFIFFWCLNTVMLVAQTFAEVEIR GEWYFMALDS I FECI YWEALLKI lALGLSYEEDEWNNLDEEIMAMAVL DFLLMQTHSFAIYHQSLFRILKVFKSLRALRAIRVLRRLSFLTSVQEVT GTLGQSLPSIAAILILMFTCLFLFSAVLRALFRKSDPKRFQNIFTTIFT LFTLLTLDDWSLI YMDSRAQGAWYI IPILVI YI I IQYFI FLNLVITVLV DSFQVTEHEEEAAIEAALLKTSQQETQERIRSIQSRMQDLTTAQYQTLT AVNTALLHLHGQDLKPFLTERSVSLANVRRISLEKKTEKSPRMF

[0164] Pt-huCatSper2 MATMQGIVENGQDKWEPEKLEDANRNQPGKRVRIMISSSLLQEKLQTP SEQ ID N0:6 ERLETVHRRDESLQGGDEEYSSHTAATTSADASSSNLSVRSVHHDEAED PSDSS FSAI PEDTAPACNEKVITSSIKSHASNERSNSEERKKEKPPTLP PSYASTIRKTRQFAGKLVNNPLFKNFIIFLVFLNTIILMVEIELLESTN TKLWPLKLTLEVAAWFILLIFILEILLKWLSNFSVFWKSAWNVFDFWT MLSLLPEVWLVGVTGQSVWLQLLRICRVLRSLKLLAQFRQIQI I ILVL VRALKSMT FLLMLLL I FFY I FAVTGVYVFSE YTRS PRQDLE YHVFFS DL PNSLVTVFILFTLDHWYALLQDVWKVPEVSRI FSS I YFILWLLLGS I I F RS I IVAMMVTNFQVTEHEEEAAIEAALLKTSQQETQERIRS IQSRMQDL TTAQYQTLTAVNTALLHLHGQDLKPFLTERSVSLANVRRISLEKKTEKS PRMF

[0165] Pt-huCatSper3 MATMQGIVENGQDKWEPEKLEDANRNQPGKRVRIMISSSLLQEKLQTP SEQ ID N0:7 ERLETVHRRDESLQGGDEEYSSHTAATTSADASSSNLSVRSVHHDEAED PSDSS FSAI PEDTAPACNEKVITSSIKSHASNERSNSEERKKEKPPTLP PSYASTIRKTRQFAGKLVNNRFFKI IMISTVTSNAFFMALWTSYDIRYR LFRLLEFSEI FFVS ICTSELSMKVYVDPINYWKNGYNLLDVI I I IVMFL PYALRQLMGKQFTYLYIADGMQSLRILKLIGYSQGIRTLITAVGQTVYT VASVLLLLFLLMYI FAILGFCLFGSPDNGDHDNWGNLAAAFFTLFSLAT VDGWTDLQKQLDNRE FALSRAFT 11 FI LLAS FI FLNMFVGVMIMHTEVT EHEEEAAIEAALLKTSQQETQERIRSIQSRMQDLTTAQYQTLTAVNTAL LHLHGQDLKPFLTERSVSLANVRRISLEKKTEKSPRMF

[0166] Pt-huCatSper4 MATMQGIVENGQDKWEPEKLEDANRNQPGKRVRIMISSSLLQEKLQTP SEQ ID N0:8 ERLETVHRRDESLQGGDEEYSSHTAATTSADASSSNLSVRSVHHDEAED PSDSS FSAI PEDTAPACNEKVITSSIKSHASNERSNSEERKKEKPPTLP PS YAS T IRKTRQFAGKLVNNPAFQLLLALLLVINAI T IALRTNS YLDQK HYELFSTIDDIVLTILLCEVLLGWLNGFWIFWKDGWNILNFIIVFILLL REFINE INI PS INYTLRALRLVHVCMAVEPLARI IRVILQSVPDMANIM VLILFFMLVFSVFGVTLFGAFVPKHFQNIQVALYTLFICITQDGWVDIY SDFQTEKREYAMEIGGAIYFTIFITIGAFIGINLFVIWTTNLEVTEHE EEAAIEAALLKTSQQETQERIRSIQSRMQDLTTAQYQTLTAVNTALLHL

[0167]

[0168] HGQDLKPFLTERSVSLANVRRISLEKKTEKSPRMF

[0169] Example 2

[0170] Molecular Cloning and Lentivirus Production

[0171] Bacterial strains

[0172] NEB® 10-0 (NEB) bacterial strains were used for molecular cloning.

[0173] Mammalian cell expression constructs Cloned DNAs encoding Odontella sinensis EUKCATA1 (OsEUKCATAl, protein ID CAMPEP O 183296650) in frame with EGFP (Helliwell, Chrachri et al. 2019) was obtained from Brownlee laboratory at University of Southampton, UK. DNAs encoding full length or the transmembrane human CatSperl, 2, 3, or 4 were cloned into phCMV3 to express C-terminal HA-tagged proteins (phCMV3-huCatSperl, 2, 3, or 4-FL or -TM) by the Chung Laboratory at Yale University School of Medicine. EukCatA-CatSper chimeric constructs in phCMV3 vector were generated using NEBuilder® HiFi DNA Assembly Kit (NEB) as following. First, OsEUKCATAl was subcloned into phCMV3 in frame with a DNA fragment encoding Thrombin cleavage site followed by mCherry, 6 repeats of HIS tag (6xHIS) and a stop codon at the upstream of HA sequences in phCMV3. Second, the transmembrane domain of OsEUKCATAl was replaced with a DNA fragment encoding the transmembrane domains of either human CatSperl, 2, 3, or 4 to finally yield phCMV3-OsCatSperl, 2, 3 or 4 in fusion with mCherry (see also FIGURES 31 and FIGURES 35-37).

[0174] Amino acid sequences of chimeric EukCatA-CatSper proteins expressed by these chimeric constructs were shown in FIGURES 3A and B and FIGURES 17-23 B. hCav3.2-CatSperl-4-3-2 chimeric construct in phCMV3 vector were generated by synthesizing two gene fragments with hanging overlap by GenScript and joined using NEBuilder® HiFi DNA Assembly Kit (NEB). The first fragment contained hCATSPERl and hCATSPER4 and the second contained hCATSPER3 and hCATSPER2, each with hCav3.2 cytoplasmic domains in-between was subcloned into phCMV3 in frame with mCherry followed by HA (see also FIGURE 39). Then the whole chimera ORF with tags was transferred to pLenti (pCW57.1 Tet-on) (see also FIGURE 40). Amino acid sequences of chimeric hCav3.2-CatSperl-4-3-2 protein expressed by these chimeric constructs were shown in FIGURE 41B.

[0175] Lentivirus production

[0176] Cloned lentiviral expression vectors (PCW57.1 Tet-on huCav3.2-huCatSper-mCherry), and lentiviral packaging (psPAX2, Addgene plasmid # 12,260) and envelop (pMD2. G, Addgene plasmid # 12,259) plasmids (gift from Didier Trono) transfected into cultured HEK293 cells using polyethylenimine (El). Culture medium was replaced with DMEM complete media (DMEM with 10% FBS and 4 mM Glutamine) 20 h after the transfection. Virus was harbvested at 48, 72 and 96 h post transfection. Collected medium with virus particles were pooled and filtered through a 0.45 mm PES filter. The viral supernatant was aliquoted, snap frozen in liquid nitrogen and stored at - 80° C until use. Cell culture

[0177] Mammalian cell lines

[0178] HEK293 cells (ATCC; derived from female embryonic kidney) or COS7 cells (ATCC; derived from African green monkey kidney) were cultured in DMEM (GIBCO) containing 10% FBS (Thermo fisher) and lx Pen / Strep (GIBCO) at 37 °C, 5% CO2 condition.

[0179] Stable cell line generation

[0180] HEK293 cells transiently transfected with plasmids phCMV3-Os-hCATSPERl, 2, 3 or 4-mCherry or pCW57.1-hCav3.2-hCATSPERl-4-3-2-mCherry were selected by 2mg / ml puromycin to establish monoclonal cells lines. For Os-hCATSPERl, 2, 3 or 4-mCherry, plasmids phCMV3-Os-hCATSPERl, 2, 3 or 4-mCherry were transiently transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen). Media was changed with fresh DMEM containing 10%FBS and 2mg / ml puromycin 72 h after transfection. Once the polyclonal populations were sufficiently expanded, single-cell suspension was prepared and seeded in a 96-well plate to establish monoclonal cells lines. For huCav3.2-huCatSper-mCherry, HEK293 cells were transduced with the lentiviral particles. Briefly, HEK293 cells were mixed with a range of lentivirus dilutions in the in DMEM complete media and incubate at 37C, 5% CO2. Meida was changed with fresh DMEM complete media with 2mg / ml puromycin 72 h after transduction. Once the polyclonal populations were sufficiently expanded, single-cell suspension was prepared and seeded in a 96-well plate to establish monoclonal cells lines.

[0181] Immunocytochemistry

[0182] Alexa Fluro 555 Conjugated Cholera Toxin Subunits B (CTxB, Invitrogen C34776) was used to label HEK293 plasma membrane (i.e. surface marker). 24 h after transfection, HEK293 cells were washed twice with pre-warmed PBS, then labeled with 2.5ug / ml CTxB containing HBSS at 37 °C for 15 min. Labelled cells were further washed with PBS and fixed with 4% formaldehyde for 15 min at room temperature. Coverslips were mounted with Prolong Gold Antifade Mount (ThermoFisher) and imaged with LSM710 using Plan-Apochrombat 63X / 1.40 objective lens (Carl Zeiss). COS7 cells were transfect with an indicated plasmids for 24 h and fixed with 4% formaldehyde for 15 min at room temperature. Fixed samples were permeabilized using 0.1% Triton X-100 in PBS at RT for 10 minutes, washed in PBS, and blocked with 3% BSA in PBS at RT for 1 h. Cells were stained with anti-HA rabbit primary antibody (CST 3724, 1: 500) at 4 °C overnight. After washing in PBS, the samples were incubated with goat anti-rabbit Alexa488 (Invitrogen, 1: 1,000) in 3% BSA in PBS at RT for 1 hr. Immunostained samples were mounted with Prolong gold (Invitrogen), followed by imaging with LSM710 using Plan-Apochrombat 63X / 1 (Carl Zeiss). Represented images of each chimeric protein expressed in HEK293 cells in FIGURE 24-30A and B, and FIGURE 42 demonstrate the plasma membrane localization of each recombinant protein.

[0183] Fluorescent plate reader Ca2+assay

[0184] HEK293 cells expressing each chimeric channel in fusion with mCherry were loaded with calcium indicators (Fluo-4 AM, Life Technologies) at 10 pM and 0.02% pluronic acid-127 (F127, Life Technologies) in DMEM without phenol red for 45 min at 37°C with 5% CO2. After loading, cells were washed twice and then incubated for a further 30 minutes with DMEM without phenol red at 37°C with 5% CO2. A fluorescent plate reader (Spark Microplate Reader, Tecan) with injector was used to measure fluorescence intensity using an excitation wavelength of 485 nm and emission wavelength of 535 nm. A baseline fluorescence recording was established for 60 seconds, then high K+ and NH4C1 DMEM solution was injected to reach a final concentration of 135 mM KC1 and recorded for another 60 seconds. Cells were maintained at 37C with 5% CO2 during recordings in the Spark microplate reader. A representative trace from HEK293 cells expressing Os-hCATSPER4, Os-hCATSPERl, Os-hCATSPER2, Os-hCATSPER3, and Cav3.2-hCATSPERl-4-3-2 are shown in FIGURE 32A, FIGURE 38 and FIGURE 43A. For HEK293 cells not expressing the chimeric proteins, 10 pM ionomycin was added at the end of imaging as shown in FIGURE 43B

[0185] Electrophysiological recording of HEK293 expressing chimeric human CatSper

[0186] The chimeric CatSper channels expressed in HEK293 cells were recorded in whole cell configuration. The standard baseline bath solution was (mM): 140 NaMeSO3, 2 CaCh, 1 MgCh, 10 D-Glucose monohydrate, 20 HEPES, pH 7.4 with NaOH. The standard pipette solution was (mM): 140 CsMeSO3, 10 CsCl, 5 EGTA, 15 HEPES, pH 7.3 with CsOH. ICatsper was measured as monovalent current in divalent-free conditions (DVF) (mM): 140 NaMeSO3, 1 EGTA, 25 HEPES, 10 D-Glucose monohydrate, pH 7.4 with NaOH. Mg2+block of IcatSper was tested in Mg2+control, nominal Ca2+free (mM): 140 NaMeSO3, 1 MgCh, 25 HEPES, 10 D-Glucose monohydrate, pH 7.4 with NaOH. Ba conductance of IcatSper was measured in Ba2+control, nominal Ca2+free (mM): 140 NaMeSO3, 1 BaCh, 25 HEPES, 10 D-Glucose monohydrate, pH 7.4 with NaOH. Represented IcatSpe ^ inward monovalent currents of Os-hCATSPER4 (FIGURES 33 and 34, orange) and hCav3.2-hCATSPERl-4-3-2 (FIGURE 44, blue) expressed in HEK293 cells upon DVF are shown.

[0187] FLIPR-based activator screen

[0188] The monoclonal stable cells (e.g., El IL) were plated at 10,000 cells / well in 384-well poly-D-lysine coated plates on day 1 and incubated for 24 hours (about 18-30 hours, often 24 hours). On day 2, cell confluency should be 95-100%. Media was removed and replaced with prewarmed HBSS (20 mM HEPES, pH 7.3). BD calcium dye was added and incubated for 30 min at 37C and then 30 min at room temperature (RT). lOx concentration of library compounds (100 uM for final 10 uM assay concentration) were diluted in HBSS intermediate plate using 0.1% DMSO as negative control and high potassium and alkalinization (135 mM KC1 and 20 mM NH4C1 final) as positive control, and ionomycin (2 uM final) as reference control. Baseline was collected for 30 seconds by FLIPR before dispensing 5 ul of compounds to 50 ul of cells and read for 400 seconds (6.8 minutes). FLIPR calculates Max-Min of this single-phase reading and gives the maximal signal which is used to flag compounds that may be fluorescent or interacting with the calcium dye leading to changes in fluorescence that are not related to the target’s activity. CatSper chimeric cell lines were counter screened against the parental HEK293 cells and Cav3.2 cell line. FIGURE 45 is shown.

[0189] FLIPR-based inhibitor screen

[0190] The monoclonal stable cells (e.g., El IL) were plated at 10,000 cells / well in 384-well poly-D-lysine coated plates on day 1 and incubated for 24 hours. On day 2, cell confluency should be 95-100%. Media was removed and replaced with pre-warmed HBSS (20 mM HEPES, pH 7.3). BD calcium dye was added and incubated for 30 min at 37C and then 30 min at RT. lOx concentration of library compounds (100 uM for final 10 uM assay concentration) were diluted in HBSS intermediate plate. Activator plate is set using high potassium and alkalinization (135 mM KC1 and 20 mM NH4C1 final) as activating condition. FLIPR is setup by a protocol signal test and adjusting gain that the RFUs across the plate are about 1,000. The FLIPR run time is 15 min. On FLIPR, baseline was collected for 20 seconds by FLIPR before dispensing 5 ul of Inhibitor (compounds) to 50 ul of cells and read for 400 seconds (6.8) minutes. During this first phase, some compounds may increase or decrease the calcium signal which may affect the detection of inhibition of activation. The wells with 0.1% DMSO (vehicle) addition instead of compounds in Phase 1 with activator addition in Phase 2 is used as negative control (no inhibition) while vehicle wells in Phase 1 with water addition (no activator) and lonomycin (2 uM final) in Phase 2 is used as positive control (full inhibition) and reference control, respectively. The Max-Min for the first phase is calculated by the FLIPR and reported as Max -Min #1. The FLIPR then adds 5.5 ul of activator and reads for 400 sec (6.8 min). The Max -Min for this second phase of activation is calculated by the FLIPR and is reported as Max -Min #2. Activity cutoffs were calculated for each phase of the calcium response. Max -Min #2 detects inhibition of activation. Max -Min #1 and Max #1 can be used to flag compounds that either increase or decrease calcium signal prior to the addition of activator. CatSper chimeric cells lines were counter screened against the parental HEK293 cells and Cav3.2 cell line. FIGURE 46 is shown.

[0191] Results from Example 2

[0192] Full-length or the transmembrane domains of human CATSPER1-4 are retained intracellularly. Previous attempts to express the full-length pore-forming (a) CATSPER subunits (1-4) in heterologous systems failed, as the proteins do not traffic to the plasma membrane and are instead retained within the ER and Golgi (Chung, Navarro et al. 2011, Chung, Miki et al. 2017) (see also FIGURE 5A and 6). To determine if the soluble domains contained retention signals, we generated truncated constructs containing only the transmembrane (TM) domains of each CATSPER subunit. These TM-only constructs also fail to traffic to the plasma membrane and showed similar intracellular retention (FIGURE 5B). These results suggest that ER / Golgi retention is an intrinsic property of the TM domains themselves and is not caused by specific retention signals in the soluble N- or C-termini.

[0193] Single-domain (ID) chimeric channels containing CatSper TM domains traffic to the plasma membrane in HEK293 cells.

[0194] EukCat channels are a family of 6-TM single-domain (ID) voltage-gated cation channels from marine phytoplankton and distantly related to NaChBac and CatSper (Helliwell, Chrachri et al. 2019). Notably, EukCatA class channels, such as OsEUKCATAl, can be functionally expressed in HEK293 cells (Helliwell, Chrachri et al. 2019). We hypothesized the soluble cytoplasmic domains of these channels contain intrinsic motifs that promote proper tetrameric assembly and trafficking to the plasma membrane. To test this, we engineered chimeric channels by fusing the 6-TM domain of each of human CATSPER a subunit (1-4) to the N-and C-terminal cytosolic domains of OsEUKCATAl (FIGURES 17, 1819, 23, 31 and 36-38). Consistent with our hypothesis, all four chimeric channels successfully trafficked to the plasma membrane of HEK293 cells, where they co-localize with the plasma membrane marker Choleratoxin B (CTxB) (FIGURES 32 and 39). This is in stark contrast to the ER / Golgi localization observed for full-length or TM-only CATSPER1-4 proteins (FIGURE 5 A and B).

[0195] ID chimeric CatSper channels are functional and recapitulate native channel properties.

[0196] To determine if these plasma membrane-localized chimeras were functional, we performed Ca imaging using the indicator Fluo-4 that enables to detect the changes in the intracellular calcium levels. Native CatSper channels in both mouse and human sperm cells are activated by membrane depolarization and intracellular alkalinization (Kirichok, Navarro et al. 2006, Lishko, Botchkina et al. 2011, Strunker, Goodwin et al. 2011). Accordingly, we stimulated the cells with a high potassium (135 mM K ) solution containing 20 mM NH4CI. Monoclonal HEK293 cell lines stably expressing the Os-hCATSPER4 exhibited a robust, stimulusdependent increase in intracellular calcium (FIGURES 32A and B and 40). In contrast, parental HEK293 cells showed no response under the same conditions (FIGURE 32B).

[0197] Furthermore, patch-clamp recording of cells expressing the Os-hCATSPER4 chimera conducted an IcatSper- Q current. This current shared key biophysical properties with native CatSper: it was carried by Na+in divalent-free (DVF) solutions, activated by intracellular alkalinization, and blocked by Mg (FIGURES 33 and 34). Together, these results provide proof-of-principle for reconstituting functional, homotetrameric human CatSper channels in a mammalian system suitable for HTS. This ID platform enables the study of each poreforming subunit in isolation, allowing for the characterization of its specific contributions to channel biophysics.

[0198] A four-domain (4D) chimeric channel mimicking the native heterotetramer is also functional.

[0199] The recent cryo-EM structure of mouse CatSper channel revealed that the four ID a-subunits (CATSPER1-4) assemble into a heterotetramer with a specific counterclockwise order (1-4- 3-2) (Lin, Ke et al. 2021). This architecture is analogous to four-domain (4D) voltage-gated channels like Cav3.2, which express functionally as a single polypeptide chain (Proft, Rzhepetskyy et al. 2017, Arteaga-Tlecuitl, Sanchez- Sandoval et al. 2018). Based on this structural insight, we designed a single-polypeptide 4D chimeric channel to mimic the native pore arrangement. We tandemly linked the human CATSPER TM domains in their native 1- 4-3-2 order, using the human Cav3.2 channel as a structural scaffold (FIGURES 41-43).

[0200] We established a monoclonal, doxycline-inducible cell line expressing the Cav3.2-hCATSPERl-4-3-2 channel (FIGURE 42). Using Ca2+imaging, we confirmed this 4D chimera was functional, observing a significant increase in intracellular Ca2+ upon stimulation with alkalinization-depolarizing solution (FIGURE 43A and B). Patch-clamp recordings further revealed an IcatSper- Q current carried (FIGURE 44). Notably, this current was not activated by progesterone (P4), a known human CatSper agonist, suggesting the P4 binding site likely resides on an auxiliary subunit absent in this minimal system. Conversely, the current was partially blocked by the non-specific CatSper inhibitor NNC 55-0396 and completely blocked by 1 mM extracellular Mg (FIGURE 44). These results demonstrate that the NNC binding site is within the tetrameric pore complex and validates the reconstitution of a functional, heterotetrameric human CatSper pore suitable for direct screening.

[0201] The ability to identify CatSper selective hit molecules by pilot activator and inhibitor screens prove the screening platform are robust to find novel CatSper modulators. We then used the monoclonal cell line expressing hCATSPER4 channel (Fig. 32) to establish a high throughput screening platform for identifying potential CatSper activators and inhibitors. Using FLIPR calcium assay, we performed pilot screens with 2,471 molecules and identified hit molecules in both activator (FIGURE 45) and inhibitor (FIGURE 46) screens. Initial hits were further screened against parental HEK293 cells and a cell line stably expressing the T-type Ca channel, Cav3.2, to find CatSper-selective modulators. These results prove these novel cell-based screening platform are robust to be used future HTS for finding novel CatSper modulators with the defined binding sites.

[0202] Discussion This study is the first to report the successful functionally expression of CatSper channels in a mammalian heterologous system, a long-standing challenge in the field. Our strategy of creating chimeric channels provides two distinct and powerful platforms for drug discovery and basic research.

[0203] The EukCat-based ID chimeras are expected for homotetramers. This platform is ideal for launching the first direct, high-throughput screens (HTS) against individual human CatSper subunits for contraceptive and pro-fertility agents. Furthermore, these isolated subunit channels will be invaluable for dissecting the specific contributions of each subunit to ion selectivity, ligand binding, and gating mechanisms. They also provide a robust system to validate and characterize low-affinity modulators previously identified from screens of native human sperm (Carlson, Burnett et al. 2009, Gruber, Johnston et al. 2020, Carlson, Georg et al. 2021). The Cav3.2-based 4D chimera generate a structure-guided heterotetrametric pore that more closely mimic the native CatSper channel, albeit in a minimal, tractable format. This system is crucial for screening and validating compounds that many require the complete heterotetrameric interface for binding.

[0204] In summary, the development and validation of these two complementary chimeric CatSper systems, one homotetrameric (ID) and one heterotetrameric (4D), provide a versatile toolkit for cell -based HTS and mechanistic studies. This work removes a critical bottleneck in the field and paves the way for the discovery and development of novel CatSper modulators for reproductive health.

[0205] TABLE 2 Amino acid sequences of chimeric CatSper channels from Example 2

[0206] Chimeric channel Amino acid sequence

[0207] Os-hCATSPERl MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID NO:1 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLLAFETFIF FWCLNTVMLVAQTFAEVEIRGEWYFMALDSIFFCIYWEALLKIIALG LSYFFDFWNNLDFFIMAMAVLDFLLMQTHSFAIYHQSLFRILKVFKSLR ALRAIRVLRRLSFLTSVQEVTGTLGQSLPSIAAILILMFTCLFLFSAVL RALFRKSDPKRFQNIFTTIFTLFTLLTLDDWSLIYMDSRAQGAWYIIPI LVIYIIIQYFIFLNLVITVLVDSFQALHEDERAKLHGTYESESEDIEDD

[0208]

[0209] EPDSEEQIKALETQIEELTRIQESTMMTLEYLTQQLQMVHLQQNQTDS

[0210] Os-hCATSPER2 MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID NO:2 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLPLFKNFII

[0211]

[0212] FLVFLNTIILMVEIELLESTNTKLWPLKLTLEVAAWFILLIFILEILLK WLSNFSVFWKSAWNVFDFWTMLSLLPEVWLVGVTGQSVWLQLLRICR VLRSLKLLAQFRQIQIIILVLVRALKSMTFLLMLLLIFFYIFAVTGVYV FSEYTRSPRQDLEYHVFFSDLPNSLVTVFILFTLDHWYALLQDVWKVPE VSRI ESS I YFILWLLLGS I I FRS I IVAMMVTNFQALHEDERAKLHGTYE SESEDIEDDEPDSEEQIKALETQIEELTRIQESTMMTLEYLTQQLQMVH

[0213]

[0214] LQQNQTDS

[0215] Os-hCATPSER3 MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID N0:3 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLRFFKI IMI STVTSNAFFMALWTSYDIRYRLFRLLEFSEIFFVSICTSELSMKVYVDP INYWKNGYNLLDVI I I IVMFLPYALRQLMGKQFTYLYIADGMQSLRILK LIGYSQGIRTLITAVGQTVYTVASVLLLLFLLMYIFAILGFCLFGSPDN GDHDNWGNLAAAFFTLFSLATVDGWTDLQKQLDNREFALSRAFTIIFIL LASFIFLNMFVGVMIMHTEALHEDERAKLHGTYESESEDIEDDEPDSEE QIKALETQIEELTRIQESTMMTLEYLTQQLQMVHLQQNQTDS

[0216] Os-hCATSPER4 MKDENSIPNSSISTSAASQAIRRSSGGSVDPSLRDPQSKPSLHPINDND SEQ ID N0:4 AGNNDSGGVSGDDSSRNSEVEEENSPQSGSSKLCRDVFWVLPAFQLLLA LLLVINAITIALRTNSYLDQKHYELFSTIDDIVLTILLCEVLLGWLNGF WIFWKDGWNILNFIIVFILLLRFFINEINIPSINYTLRALRLVHVCMAV EPLARI IRVILQSVPDMANIMVLILFFMLVFSVFGVTLFGAFVPKHFQN IQVALYTLFICITQDGWVDIYSDFQTEKREYAMEIGGAIYFTIFITIGA FIGINLFVIWTTNLEALHEDERAKLHGTYESESEDIEDDEPDSEEQIK

[0217]

[0218] ALETQIEELTRIQESTMMTLEYLTQQLQMVHLQQNQTDS hCav3.2- MTEGARAADEVRVPLGAPPPGPAALVGASPESPGAPGREAERGSELGVS hCatSperl-4-3-2 PSESPAAERGAELGADEEQRVPYPALAATVFFCLGQTTRPRSWCLRLVC SEQ ID N0:9 NPLAFETFIFFWCLNTVMLVAQTFAEVEIRGEWYFMALDSIFFCIYW EALLKI lALGLSYFFDFWNNLDFFIMAMAVLDFLLMQTHSFAIYHQSLF RILKVFKSLRALRAIRVLRRLSFLTSVQEVTGTLGQSLPSIAAILILMF TCLFLFSAVLRALFRKSDPKRFQNIFTTIFTLFTLLTLDDWSLIYMDSR AQGAWYIIPILVIYIIIQYFIFLNLVITVLVDSFQTQFSETKQRESQLM REQRARHLSNDSTLASFSEPGSCYEELLKYVGHIFRKVKRRSLRLYARW QSRWRKKVDPSAVQGQGPGHRQRRAGRHTASVHHLVYHHHHHHHHHYHF SHGSPRRPGPEPGACDTRLVRAGAPPSPPSPGRGPPDAESVHSIYHADC H I E G P QE RARVAHAAAT AAAS LRLAT GL G TMN Y P T I L P S GVG S GKG S T S PGPKGKWAGGPPGTGGHGPLSLNSPDPYEKIPHWGEHGLGQAPGHLSG LSVPCPLPSPPAGTLTCELKSCPYCTRALEDPEGELSGSESGDSDGRGV YEFTQDVRHGDRWDPTRPPRATDTPGPGPGSPQRRAQQRAAPGEPGWMG RLWVTFSGKLRRIVDSKPAFQLLLALLLVINAITIALRTNSYLDQKHYE LFSTIDDIVLTILLCEVLLGWLNGFWIFWKDGWNILNFIIVFILLLRFF INEINIPSINYTLRALRLVHVCMAVEPLARIIRVILQSVPDMANIMVLI LFFMLVFSVFGVTLFGAFVPKHFQNIQVALYTLFICITQDGWVDIYSDF QTEKREYAMEIGGAIYFTIFITIGAFIGINLFVIWTTNLEEGFQAEGD ANRSDTDEDKTSVHFEEDFHKLRELQTTELKMCSLAVTPNGHLEGRGSL SPPLIMCTAATPMPTPKSSPFLDAAPSLPDSRRGSSSSGDPPLGDQKPP ASLRSSPCAPWGPSGAWSSRRSSWSSLGRAPSLKRRGQCGERESLLSGE GKGSTDDEAEDGRAAPGPRATPLRRAESLDPRPLRPAALPPTKCRDRDG QWALPSDFFLRIDSHREDAAELDDDSEDSCCLRLHKVLEPYKPQWCRS REAWALYLFSPQNRFRVSCQKVRFFKI IMISTVTSNAFFMALWTSYDIR YRLFRLLEFSEI FFVS ICTSELSMKVYVDPINYWKNGYNLLDVI I I IVM FLPYALRQLMGKQFTYLYIADGMQSLRILKLIGYSQGIRTLITAVGQTV YTVASVLLLLFLLMYI FAILGFCLFGSPDNGDHDNWGNLAAAFFTLFSL ATVDGWTDLQKQLDNRE FALSRAFT 11 FI LLAS FI FLNMFVGVMIMHTE HYNQPKSLDEALKYPLFKNFIIFLVFLNTIILMVEIELLESTNTKLWPL KLTLEVAAWFILLIFILEILLKWLSNFSVFWKSAWNVFDFWTMLSLLP EVWLVGVTGQSVWLQLLRICRVLRSLKLLAQFRQIQI I ILVLVRALKS MTFLLMLLLI FEYI FAVTGVYVFSEYTRSPRQDLEYHVFFSDLPNSLVT VFILFTLDHWYALLQDVWKVPEVSRI ESS I YFILWLLLGS I I FRS I IVA MMVTNFQEESNKEAREDAELDAEIELEMAQGPGSARRVDADRPPLPQES PGARDAPNLVARKVSVSRMLSLPNDSYMFRPWPASAPHPRPLQEVEME TYGAGTPLGSVASVHSPPAESCASLQIPLAVSSPARSGEPLHALSPRGT ARSPSLSRLLCRQEAVHTDSLEGKIDSPRDTLDPAEPGEKTPVRPVTQG GS LQS PPRS PRPAS VRTRKHT FGQRCVS SRPAAPGGEEAEAS DPADEEV SHITSSACPWQPTAEPHGPEASPVAGGERDLRRLYSVDAQGFLDKPGRA DEQWRPSAELGSGEPGEAKAWGPEAEPALGARRKKKMSPPCISVEPPAE DEGSARPSAAEGGSTTLRRRTPSCEATPHRDSLEPTEGSGAGGDPAAKG ERWGQASCRAEHLTVPSFAFEPLDLGVPSGDPFLDGSHSVTPESRASSS

[0219]

[0220] GAIVPLEPPESEPPMPVGDPPEKRRGLYLTVPQCPLEKPGSPSATPAPG GGADDPV

[0221]

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Claims

Claims:

1. A chimeric human CatSper polypeptide channel comprising a truncated human CatSper polypeptide comprising the 6 transmembrane domains of human a-subunit of CatSper 1, CatSper 2, CatSper3 or CatSper4 fused to soluble cytoplasmic domains of a CatSper channel which exhibits surface localization.

2. The chimeric CatSper polypeptide channel according to claim 1 wherein the soluble cytoplasmic domains are cytoplasmic domains of 0. sinensis EukCatAl (OsEukCatAl) or P. tricornatum EukCatAl (PtEukCatAl).

3. The chimerica CatSper polypeptide channel according to claim 1 or 2 wherein the soluble cytoplasmic domains are the N- and C- terminal cytosolic domains of 0. sinensis EukCatAl (OsEukCatAl) or / tricornatum EukCatAl (PtEukCatAl).

4. The chimeric CatSper polypeptide channel according to claim 2 or 3 wherein the soluble cytoplasmic domains are N and C-terminal domains of OsEukCatAl.

5. The chimeric CatSper polypeptide channel according to any one of claims 1-4 wherein the transmembrane domains comprise the 6 transmembrane domains of CatSperl, CatSper2, CatSper3 or Catsper4.

6. The chimeric CatSper polypeptide channel according to claim 5 comprising the transmembrane domain of CatSperl fused with N- and C- terminal cytoplasmic domains of OsEuKCatAl (Os-hCatSperl).

7. The chimeric CatSper polypeptide channel according to claim 5 comprising the transmembrane domain of CatSper2 fused with N- and C- terminal cytoplasmic domains of OsEuKCatAl (Os-hCatSper2).

8. The chimeric CatSper polypeptide channel according to claim 5 comprising the transmembrane domain of CatSper3 fused with N- and C- terminal cytoplasmic domains of OsEuKCatAl (Os-hCatSper3).

9. The chimeric CatSper polypeptide channel according to claim 5 comprising the transmembrane domain of CatSper4 fused with N- and C- terminal cytoplasmic domains of OsEuKCatAl (Os-hCatSper4).

10. The chimeric CatSper polypeptide channel according to claim 2 or 3 wherein the soluble cytoplasmic domains areN and C-terminal domains of PtEukCatAl.

11. The chimeric CatSper polypeptide channel according to claim 10 wherein the transmembrane domains comprise the 6 transmembrane domains of CatSper 1, CatSper2, CatSper3 or Catsper4.

12. The chimeric CatSper polypeptide channel according to claim 11 comprising the transmembrane domain of CatSperl fused with N- and C- terminal cytoplasmic domains of PtEuKCatAl (Pt-hCatSperl).

13. The chimeric CatSper polypeptide channel according to claim 11 comprising the transmembrane domain of CatSper2 fused with N- and C- terminal cytoplasmic domains of PtEuKCatAl (Pt-hCatSper2).

14. The chimeric CatSper polypeptide channel according to claim 11 comprising the transmembrane domain of CatSper3 fused with N- and C- terminal cytoplasmic domains of PtEuKCatAl (Pt-hCatSper3).

15. The chimeric CatSper polypeptide channel according to claim 11 comprising the transmembrane domain of CatSper4 fused with N- and C- terminal cytoplasmic domains of PtEuKCatAl (Os-hCatSper4).

16. A chimeric CatSper polypeptide channel of SEQ ID Nos: 1-8.

17. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 1.

18. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 2.

19. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 3.

20. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 4.

21. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 5.

22. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 6.

23. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 7.

24. The chimeric CatSper polypeptide channel of claim 16 according to SEQ ID NO: 8.

25. A chimeric human CatSper polypeptide channel comprising a human CatSper polypeptide comprising the 6 transmembrane domains of human a-subunit of CatSperl, CatSper 2, CatSper3, and CatSper4 covalently linked together as a heterotetramer in the order hCatSperl-hCatSper4-hCatSper3-HCatSper4 wherein said CatSper subunits are tandemly linked (fused) human Cav3.2 channel as a structural scaffold to provide N-terminal and C-terminal soluble domains from Cav3.2 at the end of each CatSper subunit.

26. The chimeric CatSper polypeptide channel of claim 25 according SEQ ID NO: 9.

27. The chimeric CatSper polypeptide channel according to any one of claims 1-26 further comprising a reporter protein.

28. The chimeric CatSper polypeptide channel according to claim 27 wherein said reporter protein is EFGP or mCherry.

29. The chimeric CatSper polypeptide channel according to claim 28 wherein said reporter protein is mCherry.

30. A plasmid vector which expresses a chimeric CatSper polypeptide channel according to any one of claims 1-29.

31. A plasmid vector which expresses a chimeric CatSper polypeptide channel according to either of claims 1 or claim 25.

32. A plasmid vector as set forth in any one of Figs. 1, 10-16, 31, 35-37 and 39-40, hereof.

33. A bacteria comprising a plasmid vector according to any of claims 30-32.

34. A mammalian cell comprising a plasmid vector according to any of claims 30-32.

35. The mammalian cell according to claim 35 which is a HEK293 cell or a Cos7 cell.

36. The mammalian cell according to claim 34 or 35 which a monoclonal stable transfected cell.

37. A method of expressing a chimeric CatSper polypeptide channel on the surface of a mammalian cell comprising introducing a plasmid according to any one of claims 1-32 into said cell and allowing expression of said chimeric CatSper polypeptide channel within said cell, wherein said polypeptide channel will localize on the surface of said cell.

38. A mammalian cell which expresses chimeric CatSper polypeptide channel on its surface.

39. The mammalian cell which is a stable transfected cell.

40. The mammalian cell according to claim 38 or 39 which is HEK293 or Cos7.

41. The mammalian cell according to claim any one of claims 38-40 wherein said chimeric CatSper polypeptide channel is the channel of any one of claims 1-29.

42. A method of identifying a compound of unknown CatSper Channel activity as an inhibitor or an activator / enhancer of CatSper activity, comprising providing stable, transfected cells which express a chimeric CatSper channel according to any one of claims34-36 at the surface of the cells and determining the impact of exposure of said compound on calcium flow through the chimeric CatSper channels expressed by said cells, wherein a compound which is shown to inhibit calcium flow through the CatSper channel is identified as a potential contraceptive agent and wherein a compound which is shown to increase or enhance calcium flow through the CatSper channel is identified as a potential fertility enhancing agent.

43. A method of identifying a compound of unknown activity as a potential fertility agent (enhancer of CatSper channel activity) or a potential contraceptive comprising:plating a population of stable transfected cells according to any one or 34-36 in cell media; removing the cell media from the incubated cells and replacing the cell media with buffer solution;adding calcium dye to the cells and incubating the cells;diluting a concentration of compounds of unknown activity are diluted in buffer serum which includes 0.1% DMSO as negative control, high potassium and alkalinization as positive control and 2 pM ionomycin as reference control;establishing baseline for several minutes before a concentration of compound is dispensed into cells (e.g. 5pL of drug to 50pL of cells) and recorded for at least 5-10 minutes;a maximum of calcium efflux is determined for each compound tested and provides a calcium signal which is used to flag tested compounds which have potential as fertility agents, wherein a compound which evidences a positive impact on calcium flow (increase in calcium flow) is considered a potential fertility agent and a compound which evidences negative impact on calcium flow is considered a potential contraceptive agent.

44. The method according to claim 42 wherein said assay is a Flipr assay.

45. A method of identifying a compound of unknown activity as a potential contraceptive or fertility agent (inhibitor or enhancer) of CatrSper channel activity comprising the steps of:plating a population of stable transfected cells according to any one of claims 34-36 and incubating these cells in cell media;removing the cell media from the incubated cells and replacing the cell media with buffer solution (e.g. Hanks buffered saline solution HBSS);adding calcium dye to the cells and incubating the cells in the buffered saline solution;providing a concentration of compounds of unknown activity which are diluted in buffer serum which includes DMSO as positive control (no activation), high potassium and alkalinization (as negative control) and ionomycin as reference control;establishing a baseline for each compound of unknown activity before a concentration of each compound is dispensed into cells and recorded;determining a maximum of calcium efflux for each compound tested and then adding activator which provides a calcium signal which is used to flag tested compounds which have potential as fertility or contraceptive agents, wherein a compound which increases calcium flow in the assay prior to the addition of activator is identified as a potential fertility agent and a compound which decreases / inhibits calcium flow prior to the addition of activator is considered a potential / likely contraceptive agent.

46. The method according to claim 45 wherein said assay is a Flipr assay.

47. A method of identifying a compound of unknown CatSper channel activity as an inhibitor or an activator / enhancer of CatSper activity, comprising providing stable, transfected cells which express a chimeric CatSper channel at the surface of the cells according to any one of claims 34-36, establishing a stable, resting calcium flow through said CatSper channel and determining the impact of exposure of said cells to said compound on calcium flow through the chimeric CatSper channels expressed by said cells, wherein a compound which is shown to inhibit calcium flow through the CatSper channel is identified as a potential contraceptive agent and wherein a compound which is shown to increase or enhance calcium flow through the CatSper channel is identified as a potential fertility / enhancing agent.

48. The method of claim 47 comprising comparing the inhibition or enhancement of calcium flow through said CatSper channel by said compound with a standard of known activity and determining whether said compound is identified with potential contraceptive or fertility enhancement activity.

49. The method of claim 48, wherein the standard is a known inhibitor of CatSper channel calcium flow.

50. The method of claim 48, where the standard is a known fertility enhancer of CatSper channel calcium flow.

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