NALCN channel proteins and their modulators

Abstract

The present disclosure relates, inter alia, to a complex of Na+ leak channel nonselective protein (NALCN), FAM155A (also called FAM155; family 155 member A with sequence similarity), UNC79 (uncooperative 79) and UNC80 (uncooperative 80), methods for screening molecules that regulate the activity of the complex, and identified modulators thereof.

Description

[Technical field]

[0001] Field The present disclosure relates, inter alia, to a complex of Na+ leak channel non-selective protein (NALCN), FAM155A (also called FAM155; family 155 member A with sequence similarity), UNC79 (uncooperative 79) and UNC80 (uncooperative 80), methods of screening for molecules that regulate the activity of the complex, and identified modulators thereof. [Background technology]

[0002] background In many neurons, the NALCN (Na+ leak channel nonselective) channelosome mediates the basal Na+ channel that regulates resting membrane potential, spontaneous firing, and pacemaker activity. + NALCN represents a distinct branch of the four-domain ion channel superfamily and is an orphan gene in humans. It is distantly related to the voltage-gated sodium (Na V ) channels and calcium (Ca V Unlike the canonical Na channel, NALCN is not gated by changes in membrane potential. Robust activity in heterologous systems requires the co-expression of three other proteins: FAM155A (also called FAM155; family 155 member A with sequence similarity), UNC79 (uncoordinated 79), and UNC80 (uncoordinated 80). Mutations in NALCN and UNC80 have been shown to cause a range of neurodevelopmental disorders, including, for example, motor, psychiatric, and visual disorders. FAM155A, UNC79, and UNC80 are involved in the regulation of canonical Na V or Ca V It shares no sequence similarity with the channel accessory subunits, suggesting that it may play a specialized scaffolding or functional role.

[0003] Recently, the basic structure of the NALCN-FAM155A pore-forming subcomplex was determined, revealing that FAM155A forms an integrated dome-like structure across the NALCN-selective filter, whereas the S6 gate is closed in the absence of UNC79 and UNC80. (M. Kschonsak et al., Nature 587:313-318 (2020); Y. Kang et al., Nature Comm. 11:6199 [doi.org / 10.1038 / s41467-020-20002-9] (2000); J. Xie et al., Nature Comm. 11:5831 [doi.org / 10.1038 / s41467-020-19667-z] (2000).) UNC79 and UNC80 have been proposed to physically interact with NALCN, and UNC79 has been suggested to bind to UNC80 to promote dendritic localization of NALCN. Previous attempts to form a complex of NALCN with FAM155A that further contains UNC79 and UNC80 were unsuccessful, resulting in only a NALCN-FAM155A subcomplex. (See ibid.) Summary of the Invention

[0004] overview The inventors have now succeeded in preparing, in some embodiments, complexes of all four of NALCN, FAM155A, UNC79 and UNC80 with calmodulin (CaM), as described in more detail in the Examples section below. The inventors have also determined the overall structure of this complex at 3.3-3.1 angstrom resolution by cryo-electron microscopy. The ability to obtain complexes of these proteins suitable for functional assays, such as ion channel assays and binding affinity assays, allows, inter alia, the screening of molecules to identify modulators of the complex and molecules that bind to the complex.

[0005] Thus, the present application includes, for example, a method for identifying a modulator of a complex of human NALCN (Na+ leak channel nonselective), FAM155 (family 155 member A with sequence similarity), UNC79 (uncooperative 79) and UNC80 (uncooperative 80) (human NALCN-FAM155-UNC79-UNC80 complex), comprising: (a) providing a human NALCN-FAM155-UNC79-UNC80 complex in vitro; (b) contacting the complex with a potential modulator of the complex; (c) performing an ion channel assay of the complex in the presence of the potential modulator; and (d) identifying the potential modulator as a modulator of the complex if the activity of the complex in the assay in the presence of the potential modulator is higher or lower than the activity of the complex in the assay in the absence of the potential modulator. In some examples, the ion channel assay is a patch clamp or automated patch clamp assay, an ion flux assay, or an ion or voltage sensitive dye assay. In some examples, the activity of the complex in the assay in the presence of a potential modulator is less than the activity of the complex in the assay in the absence of the potential modulator (i.e., the potential modulator reduces the activity of the complex in the assay). In some examples, the potential modulator identified in part (c) reduces the activity of the complex in the assay by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the activity of the complex in the assay in the presence of a potential modulator is greater than the activity of the complex in the assay in the absence of the potential modulator (i.e., the potential modulator increases the activity of the complex in the assay).In some embodiments, the method further includes determining the binding affinity of the potential modulator identified in part (d) to human NALCN-FAM155-UNC79-UNC80 complex, human NALCN-FAM155 complex, UNC79-UNC80 complex, or one or more of human NALCN, FAM155, UNC79, or UNC80. In some examples, the potential modulator binds to human NALCN-FAM155-UNC79-UNC80 complex with an EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less. In some embodiments, the method is carried out in the presence of a NALCN-DII-DIII linker peptide or a NALCN-DI-DII linker peptide, or an identified modulator of the human NALCN-FAM155-UNC79-UNC80 complex. In some embodiments, the linker peptide comprises the amino acid sequence of any one of SEQ ID NOs: 16-24, or the amino acid sequence of any one of SEQ ID NOs: 16-18 or 23-24. In some examples, the method further comprises determining whether the potential modulator modulates the activity of the human NALCN-FAM155-UNC79-UNC80 complex, the complex comprising a mutant human NALCN. In some examples, the mutant human NALCN comprises a substitution, insertion or deletion in one or both of the DI-DII linker or DII-DIII linker. In some examples, the DI-DII linker or DII-DIII linker is a human or mammalian Na. V or Ca V Proteins, such as human Na V1.4; contains a deletion; or contains an insertion of a GGGS element. In some examples, the mutant human NALCN comprises the amino acid sequence of any one of SEQ ID NOs: 7, 8, 12-15, 32-52, or 59-63. In some embodiments, the method further comprises determining whether the potential modulator modulates the activity of a human NALCN-FAM155-UNC79-UNC80 complex, the complex comprising a mutant human UNC79 comprising, for example, the amino acid sequence of any one of SEQ ID NOs: 53, 54, 55, 58, or 64-73. In some embodiments, the method further comprises determining whether the potential modulator modulates the activity of a human NALCN-FAM155-UNC79-UNC80 complex, the complex comprising a mutant human UNC80 comprising, for example, the amino acid sequence of any one of SEQ ID NOs: 74-85.

[0006] The present disclosure also includes a method for identifying a molecule that binds to a complex of, for example, human NALCN (Na+ leak channel nonselective), human FAM155 (family 155 member A with sequence similarity), human UNC79 (uncooperative 79) and human UNC80 (uncooperative 80): (human NALCN-FAM155-UNC79-UNC80 complex), comprising: (a) providing a human NALCN-FAM155-UNC79-UNC80 complex in vitro; (b) contacting the complex with one or more test molecules and separating bound test molecules from unbound test molecules; and (c) identifying the test molecule as a molecule that binds to the human NALCN-FAM155-UNC79-UNC80 complex if the test molecule remains bound to the complex after separating the bound test molecules from the unbound test molecules.

[0007] The disclosure also provides a method for identifying a molecule that binds to a complex of, for example, human NALCN (Na+ leak channel nonselective), human FAM155 (family 155 member A with sequence similarity), human UNC79 (uncooperative 79) and human UNC80 (uncooperative 80): (human NALCN-FAM155-UNC79-UNC80 complex), comprising: (a) providing in vitro a human NALCN-FAM155-UNC79-UNC80 complex; (b) coupling said complex to a NAL and (c) contacting the test molecule with a CN-DII-DIII linker peptide or a NALCN-DI-DII linker peptide and further contacting the test molecule with a test molecule; and (c) identifying the test molecule as a molecule that binds to the human NALCN-FAM155-UNC79-UNC80 complex if the test molecule competes with the NALCN-DII-DIII linker peptide and / or the NALCN-DI-DII linker peptide for binding to the complex.

[0008] The present disclosure also includes a method for identifying a molecule that modulates the in vitro formation or stability of a complex of, for example, human NALCN (Na+ leak channel nonselective), human FAM155 (family 155 member A with sequence similarity), human UNC79 (uncooperative 79) and human UNC80 (uncooperative 80): (human NALCN-FAM155-UNC79-UNC80 complex), comprising: (a) providing human NALCN, human FAM155, human UNC79 and human UNC80 under conditions that promote human NALCN-FAM155-UNC79-UNC80 complex formation in vitro; (b) contacting the complex with a test molecule; and (c) identifying the test molecule as a molecule that modulates the formation or stability of the human NALCN-FAM155-UNC79-UNC80 complex if the extent of complex formation in the presence of the test molecule is increased or decreased compared to the absence of the test molecule.

[0009] In some embodiments of the above methods, the potential modulator binds to the human NALCN-FAM155-UNC79-UNC80 complex with an EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less. In some embodiments, the binding affinity is determined by ELISA, AlphaLISA, or FRET assay.

[0010] In some examples, in any of the methods described herein, at least one of the human NALCN, human FAM155, human UNC79, or human UNC80 of the human NALCN-FAM155-UNC79-UNC80 complex is labeled. In some examples, at least one of the human NALCN, human FAM155, human UNC79, or human UNC80 of the human NALCN-FAM155-UNC79-UNC80 complex is bound to a matrix such as a bead, chip, or plate. In some examples, the human NALCN-FAM155-UNC79-UNC80 complex is solubilized in a lipid bilayer, detergent, or lipid nanodisc. In some examples, the method further includes determining whether a molecule binds to the human NALCN-FAM155 complex, the human UNC79-UNC80 complex, or one or more of the human NALCN, FAM155, UNC79, or UNC80.

[0011] In some embodiments, in any of the methods described herein, the method further comprises determining whether the test molecule binds to a human NALCN-FAM155-UNC79-UNC80 complex, the complex comprising a mutant human NALCN. In some examples, the mutant human NALCN comprises a substitution, insertion or deletion in one or both of the DI-DII linker or the DII-DIII linker. In some examples, the DI-DII linker or the DII-DIII linker comprises a human or mammalian Na V or Ca V Proteins, such as human Na V1.4; contains a deletion; or contains an insertion of a GGGS element (SEQ ID NO: 86). In some examples, the mutant human NALCN comprises the amino acid sequence of any one of SEQ ID NOs: 7, 8, 12-15, 32-52, or 59-63. In some examples, in any of the methods described herein, the human NALCN-FAM155-UNC79-UNC80 complex further comprises human calmodulin.

[0012] In some examples, in any of the methods described herein, the potential modulator or test molecule is a peptide, a macrocyclic polymer, or an antibody. In some examples, the potential modulator or test molecule is a small molecule.

[0013] The disclosure also includes molecules identified, for example, by the methods described herein. In some examples, such molecules are peptides, macrocyclic polymers, or antibodies. In other examples, such molecules are small molecules. The disclosure also includes identified modulators of complexes as described herein, for example, identified peptide modulators of human NALCN, for example, peptides comprising the amino acid sequence of any one of SEQ ID NOs: 16-18 or 23-24.

[0014] The disclosure also includes, for example, the use of a molecule described herein or an identified modulator in treating a neurodevelopmental disorder, a circadian rhythm disorder, or pain in a subject, as well as a method of treating a subject having a neurodevelopmental disorder, a circadian rhythm disorder, or pain, comprising, for example, administering an effective amount of a molecule described herein or an identified peptide modulator.

[0015] The disclosure includes kits that include, for example, an identified modulator of the human NALCN-FAM155-UNC79-UNC80 complex, one or more reagents for performing an ion channel assay, at least one of a labeled NALCN, UNC79, UNC80 or FAM155, which may be bound to a matrix such as a bead, chip or plate, and instructions for use.

[0016] The present disclosure also includes an isolated complex of human NALCN, UNC79, UNC80 and FAM155, which may be contained within, for example, a lipid bilayer or lipid nanodisc, and at least one of said human NALCN, UNC79, UNC80 and FAM155 may include a label or may be bound to a matrix such as a bead, plate or chip. In some examples, the complex further includes a modulator of human NALCN or a molecule identified by any of the methods herein.

[0017] Additional objects and advantages will be set forth in part in the following description and in part will be understood from the description or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0018] [Figure 1] Figures 1A-1F show the overall structure of the human NALCN channelosome. Figure 1A shows current traces from Xenopus oocytes expressing NALCN alone or with the indicated combinations of FAM155A, UNC79 and / or UNC80. NALCN subunits are wild type or contain C-terminal GFP-Flag tag fusions as indicated. In ND96 recording solution, from +80 to -100 mV, in 20 mV steps. Figure 1B shows an overall cryo-EM map. Figure 1C shows a cartoon-style representation of the entire NALCN channelosome model. Figures 1D-1F show another view of the NALCN channelosome.

[0019] [Diagram 2] Figures 2A-T show the overall structure of the UNC79-UNC80 subcomplex. Figure 2A shows a cartoon-style cylindrical representation of UNC79 and UNC80. Figure 2B shows a close-up of the NC interface. Figure 2C shows a close-up of the CN interface. Figure 2D shows a close-up of the crossover interface. Figure 2E shows the mapping of deletion and disease mutations. Figures 2F-H show a depiction of the contacts (Figure 2F), electrostatics (Figure 2G), and conserved surfaces (Figure 2H) of the crossover interface. Figures 2I-J show the structural superposition of UNC79 and UNC80. Figure 2K shows the Ub-like insertion domain of UNC80, shown in pink. Figure 2L shows the superposition of the UNC80 Ub-like domain with Ub. Figures 2M-N show the superposition of importin-α3 onto UC79. Figure 2O-P show an overlay of importin β (Figure 2O) and PP2A (Figure 2P) onto UNC79. Figure 2Q shows current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, subunits either wild type or truncated as indicated. Asterisks (*) indicate constructs containing C-terminal GFP-Flag tag fusions. From +80 to -100 mV in 20 mV steps in ND96 recording solution. Figure 2R-T show similar traces as in Figure 2Q, but with full-length N-terminally truncated (UNC79468-2635* and UNC80734-3258*) or C-terminally truncated (UNC791-2400* and UNC801-2794) constructs expressed as is or in the combinations indicated. The summary on the right shows the mean current amplitudes evoked at +80 mV from a holding potential of 0 mV for the indicated construct combinations.

[0020] [Diagram 3]Figures 3A-J show NALCN interaction with UNC79-UNC80 subcomplex. Figure 3A shows NALCN in cartoon representation and UNC79 and UNC80 in surface representation. Figure 3B shows a full view of the NALCN DII-DIII linker structure. Figure 3C shows a close-up of the DI-DII linker-UNC79 interaction interface. Figure 3D shows a diagram of the hinge module. Figure 3E shows a close-up of the DII-DIII linker lasso loop interaction with UNC80. Figure 3F shows a close-up of the DII-DIII linker α2-helix interaction with UNC80. Figure 3G shows a diagram of the DII-DIII linker Arg717 interaction with UNC79 and UNC80. Figure 3H shows a diagram of the NALCN CTD and CaM interaction with UNC80. Figure 3I-J show current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunits are wild type or deleted, in ND96 recording solution from +80 to -100 mV, in 20 mV steps. The summary on the right shows the average current amplitudes evoked at +80 mV from a holding potential of 0 mV for the indicated combinations of constructs.

[0021] [Figure 4]Figure 4A-4HF show the pore structure and gating model of the NALCN channelosome. Figure 4A shows the pore radius of the ion conduction pathway in the NALCN channelosome or NALCN-FAM155A subcomplex structure. Figure 4B shows the superimposed, subcellular view highlighting the S6 gate region in the NALCN channelosome and NALCN-FAM155A subcomplex structures. Figure 4C is a top view showing the superimposition of two conformations of the NALCN channelosome, with the NALCN subunits removed for clarity. The inset highlights the DI-DII and DII-DIII linkers, which are both cyan or pink in their respective conformations. Figure 4D shows a schematic of NALCN showing where the linker engineering was performed. Figure 4E shows the current traces (top left panel) and average current amplitudes (bottom left panel) of constructs WT and A-J (right panel). The top left panel of Figure 4E shows exemplary current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where NALCN is shown to be wild type or engineered. The bottom left panel of Figure 4E shows a summary of the mean current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV expressing the indicated construct combinations. Figure 4F shows current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where control (HO) or the indicated isolated DII-DIII linker constructs are expressed in trans. Figure 4G shows a schematic of the DII-DIII linker (top panel) and a summary showing the mean current amplitudes evoked at +80 mV from a holding potential of 0 mV for the indicated construct combinations (bottom panel). Figure 4H shows a schematic of a potential gating model, shown either throughout the NALCN channelosome (top) or at the level of the S6 gate.

[0022] [Diagram 5]Figures 5A-5J illustrate the purification and structure determination of the NALCN channelosome. Figure 5A shows the expression and purification scheme of the NALCN channelosome protein. Figures 5B-5C show exemplary size-exclusion chromatographs (Figure 5B) and SDS-PAGE (Figure 5C) of nanodisc-reconstructed NALCN channelosome samples. Figure 5D shows exemplary cryo-EM micrograph images of the NALCN channelosome-MSP1E3D1 complex. Figure 5E shows representative 2D class averages after two rounds of 2D classification from 200 classes and approximately 720,000 particles. Figure 5F shows the data collection and processing workflow. Figure 5G shows conformation 1, where FSC between the two half datasets results in an overall resolution estimate of approximately 3.1 Å resolution. Figure 5H shows conformation 1, a heatmap display of the distribution of assigned particle orientations. Figure 5I shows conformation 2, where FSC between the two half data sets yields an overall resolution estimate of approximately 3.1 A resolution. Figure 5J shows conformation 2, a heatmap depiction of the distribution of assigned particle orientations.

[0023] [Figure 6] Figures 6A-6D show selected cryo-EM map regions of the NALCN channelosome, with exemplary 3D map overlays for the DI-DII linker (Figure 6A), the DII-DIII linker (Figure 6B), the NALCN-CTD (Figure 6C), and CaM (Figure 6D).

[0024] [Figure 7] Figures 7A-7B show the structures of NALCN and FAM155A in the NALCN channelosome and the NALCN-FAM155A subcomplex. Figure 7A shows a comparison of the NALCN subunits in the NALCN-FAM155A subcomplex and the NALCN channelosome (UNC79 and UNC80 have been removed for clarity). Figure 7B shows a superposition of the FAM155A subunit in the NALCN-FAM155A subcomplex and the NALCN channelosome.

[0025] [Figure 8] Figures 8A-8B show that UNC79 and UNC80 are HEAT repeat proteins. Figure 8A shows a side view and a top view of UNC79. The positions of irregular loops that are more than 50 residues in length are indicated. Figure 8B shows a side view and a top view of UNC80. The positions of irregular loops that are more than 50 residues in length are indicated.

[0026] [Figure 9] FIG. 9 shows a multiple sequence alignment of UNC79.

[0027] [Figure 10] FIG. 10 shows a multiple sequence alignment of UNC80.

[0028] [Figure 11]Figures 11A-11O show structure-function analysis of UNC79 and UNC80. Figures 11A and 11D show schematics of UNC79 (Figure 11A) and UNC80 (Figure 11D), with the corresponding amino acid residues of the fragments, respectively. Figures 11C and 11E show exemplary current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where various isolated ~500 residue fragments of either UNC79 or UNC80 are co-expressed in trans at +80 to -80 mV in 40 mV increments in ND96 recording solution. Figure 11C and Figure 11F show a summary of the average current amplitudes evoked at +80 mV (top bar graphs in Figure 11C and Figure 11F) or -80 mV (bottom bar graphs in Figure 11C and Figure 11F) from a holding potential of 0 mV for the indicated combinations of constructs. Figure 11G shows a schematic of UNC80 with the corresponding truncated constructs shown. Figure 11H shows exemplary current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where wild-type UNC80 or truncated constructs are expressed in ND96 recording solution from +80 to -80 mV, in 20 mV increments. Asterisks (*) indicate constructs containing a fusion of a C-terminal GFP-Flag tag. Figure 11I shows a summary of the average current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated construct combinations. Figure 11J shows a schematic diagram of UNC80 with the corresponding nonsense mutant constructs shown. Figure 11K shows exemplary current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where wild-type UNC80 or nonsense mutant constructs are expressed in ND96 recording solution at +80 to -80 mV, in 20 mV increments. Figure 11L shows a summary of the average current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated construct combinations.Figure 11M shows exemplary current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, with wild-type UNC80 or missense mutant constructs expressed from +80 to -80 mV, in 20 mV increments, in ND96 recording solution. Figure 11N shows a summary of the average current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated combinations of constructs. Figure 11O shows a zoomed-in view of select missense mutations previously identified in UNC80 mapped onto the UNC79-UNC80 subcomplex structure.

[0029] [Figure 12] Figures 12A-G show characterization of the NALCN-NaV1.4 chimera. The top panels of Figures 12A-F show schematics of human NALCN and human NALCN-rat NaV1.4 chimeric constructs. The bottom panels of Figures 12A-F show exemplary current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, with wild-type or chimeric truncated NALCN constructs expressed from +80 to -80 mV in 20 mV increments in ND96 recording solution. Figure 12G shows a summary of the mean current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated constructs.

[0030] [Figure 13]Figures 13A-C show structure-function of the C-terminal domain of NALCN. Figure 13A shows current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunits are wild type or exhibit CTD deletions or mutations. From +80 to -100 mV, in 20 mV steps, in ND96 recording solution. Figure 13B shows a summary of the mean current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated constructs. Figure 13C shows current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79, UNC80 and control (HO) or isolated NALCN-CTD constructs in trans. in ND96 recording solution, from +80 to -100 mV, in 20 mV steps. Figure 13D shows a summary of the average current amplitudes evoked at +80 mV from a holding potential of 0 mV in the indicated conditions. Figure 13E shows current traces from Xenopus oocytes expressing wild-type NALCN (1-1738) or the indicated C-terminal truncated constructs, recorded in ND96 (top) and divalent cation (X2+)-free buffer (bottom). from +80 to -100 mV, in 20 mV steps. Figure 13F shows the fold increase in inward current evoked at -100 mV for wild-type NALCN and truncated mutants in response to removal of divalent cations.

[0031] [Figure 14]Figures 14A-D show structure-function of NALCN DI-DII and DII-DIII linkers. Figure 14A shows current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunits are wild type or show insertions, deletions or mutations in the DI-DII linker. From +80 to -100 mV in 20 mV steps in ND96 recording solution. Figure 14B shows a summary of the average current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated constructs. Figure 14C shows current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunits are wild type or show insertions, deletions or mutations in the DII-DIII linker. in ND96 recording solution, from +80 to -100 mV, in 20 mV steps. Figure 14D shows a summary of the mean current amplitudes evoked at +80 mV (upper bar graph) or -80 mV (lower bar graph) from a holding potential of 0 mV for the indicated constructs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description of Specific Embodiments definition As used herein, the term about refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly stated. The term about generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values ​​or ranges, those terms modify all of the values ​​or ranges provided in the list. In some instances, the term about may include numerical values ​​that are rounded to the nearest significant figure.

[0033] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The following terms, as utilized in accordance with the present disclosure, shall be understood to have the following meanings, unless otherwise indicated:

[0034] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of a multiple dependent claim, the use of "or" refers only to any one or more of the preceding independent or dependent claims. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and element, and components that contain more than one subunit, unless specifically stated otherwise.

[0035] The transitional term "consisting essentially of" as used herein, when referring to steps of a claimed process, means that the process does not contain additional steps beyond the specified steps that would materially affect the basic and novel characteristics of the process. The transitional term "consisting essentially of" as used herein, when referring to a composition or article of manufacture, e.g., a kit, means that the process does not contain additional components beyond the specified components that would materially affect its basic and novel characteristics.

[0036] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include every integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0038] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure that can be had by reference to the entire specification. Thus, the terms defined below are more fully defined by reference to the entire specification.

[0039] All proteins described herein are human proteins unless expressly stated otherwise, such as by phrases such as "mammalian NALCN" or "mouse NALCN."

[0040] The term "NALCN" refers to human Na+ leak channel nonselective protein. An exemplary amino acid sequence of the protein is provided in SEQ ID NO: 1 and can be found in the UniProt database under accession number Q8IZF0. This protein is also referred to as VGCNL1 (voltage-gated channel-like protein 1).

[0041] "FAM155" or "FAM155A" protein refers to the human family with sequence similarity 155 member A protein, a transmembrane protein that interacts with NALCN. An exemplary amino acid sequence of the protein can be found in SEQ ID NO: 2 or in the UniProt database under accession number B1AL88.

[0042] "UNC79" protein refers to human "uncooperative 79" protein or human "protein unc-79 homolog". This protein is produced by transcription of the human UNC79 or KIAA1409 gene. An exemplary amino acid sequence can be found in SEQ ID NO: 3 or in the UniProt database under accession number Q9P2D8.

[0043] "UNC80" protein refers to human "uncooperative 80" protein or human "protein unc-80 homologue". This protein is produced by transcription of the human UNC80 or KIAA1843 or C2orf21 gene. An exemplary amino acid sequence can be found in SEQ ID NO: 4 or in the UniProt database under accession number Q8N2C7.

[0044] The term "CaM" or "calmodulin" refers to the human calmodulin protein, which is also abbreviated as CALM, CALM1, or CAM1. An exemplary amino acid sequence is provided in SEQ ID NO:5 or can be found in the UniProt database under accession number P0DP23.

[0045] As used herein, "mutant NALCN" refers to a human NALCN protein that contains at least one engineered amino acid substitution, insertion or deletion compared to the protein of SEQ ID NO: 1. In some examples, "mutant NALCN" refers to a human NALCN protein that contains at least one engineered amino acid substitution, insertion or deletion compared to the protein of SEQ ID NO: 1. V or Ca V In some instances, the region or complete domain of the various ion channel proteins includes at least one amino acid substitution that includes at least one equivalent / corresponding residue of a Na channel protein. V or Ca V The equivalent region or domain of various ion channel proteins, such as family proteins, is replaced with part or all of the DI-DII or DII-DIII linker. In such examples, the mutant NALCN protein contains amino acid sequence segments from two or more natural proteins, and therefore may be alternatively referred to herein as a "chimeric NALCN" or "chimera."

[0046] The term "chimeric" as used herein, when referring to a protein, means that the protein is composed of amino acid sequences from two or more naturally occurring proteins. For example, as described above, a "chimeric NALCN" as used herein refers to a protein in which at least one region of the NALCN protein is a region derived from a human Na V or Ca V Refers to a type of mutant NALCN that has been exchanged with a corresponding region from a different ion channel protein, such as a family member protein. In some instances, the exchanged region may be a complete domain, or one or more individual transmembrane alpha helices or linker regions or loops within the domain, or a portion of an alpha helix and / or loop within the domain.

[0047] As used herein, the terms "corresponding" or "equivalent" are used interchangeably to refer to residues or regions from one protein that replace residues or regions deleted from a different protein. As used herein, "corresponding" or "equivalent" residues or regions are residues or regions that are in the same position in two proteins when properly folded. In some instances, such corresponding or equivalent regions or amino acid residues can be identified using sequence alignment and structural information of the two proteins.

[0048] As used herein, a "modulator" refers to a molecule that can change the behavior of a protein or a complex of proteins, such as NALCN alone or a complex of FAM155, UNC79, and UNC80. For example, in some cases, a modulator can change the behavior of a target protein or complex by binding to the protein. A modulator herein can act to increase or decrease the activity of a protein, such as, for example, the degree to which the protein regulates the flow of ions across a cell membrane. A modulator that decreases the activity of NALCN is, for example, an "inhibitor" of NALCN. A modulator that increases the activity of NALCN is, for example, an "activator" of NALCN. In some embodiments, a modulator can modulate the activity of NALCN only under certain conditions, such as in the presence of certain ions, or when NALCN is complexed with other proteins, such as FAM155, UNC79, and UNC80. A molecule that is a "modulator" of the NALCN-FAM155-UNC79-UNC80 complex increases or decreases the activity of the complex, for example, in an ion channel assay. Such molecules can affect the function of the complex through a variety of mechanisms, for example by affecting the binding, folding or stability between members of the complex, or the trafficking or localization of one of the members of the complex, or by affecting the folding or stability of an interface between proteins, or by blocking the entry or exit of molecules from the central channel of the complex.

[0049] For purposes of this specification, "NALCN-FAM155-UNC79-UNC80 complex" and "NALCN complex" are used interchangeably unless it is made clear that they are different.

[0050] As used herein, a "potential modulator" of a NALCN complex is a molecule that is tested to determine whether it acts as a modulator of the NALCN complex.

[0051] "Ion channel assay" herein refers to an assay used to measure the activity of an ion channel protein, such as a voltage-gated sodium channel. A variety of assays and assay formats are commonly used in the art, and many are provided in an automated format for high-throughput screening (HTS) analysis. Examples include ion flux assays, e.g., using radioactive ions, e.g., radioactive Na+ ions, ion-sensitive or voltage-sensitive dye assays, e.g., fluorescent assays, e.g., using fluorescent indicator molecules in which the fluorescent signal increases or decreases with changes in ion concentration, and various types of patch clamp assays. Such assays may directly or indirectly measure changes in ion current across a membrane containing an ion channel protein under various conditions. In some examples, such assays are used to evaluate the "activity" of an ion channel protein in the presence or absence of a potential or identified modulator. The term "activity" in this sense is meant in its broadest sense, given that these various assays directly or indirectly measure the activity of a protein through the measurement of various parameters, such as fluorescence, radioactivity, or changes in ion current.

[0052] "Patch clamp assay" is used herein in the broadest sense to refer to an assay used to assess changes in the movement of ions across a small patch of cell membrane that contains, for example, ion channel proteins under a variety of solution conditions.

[0053] As used herein, the term "peptide" refers to a chain of 50 or fewer amino acids linked by peptide bonds, including amino acid chains of 2-50, 2-15, 2-10, 2-8, or 6-14 amino acids.

[0054] As used herein, the term "small molecule" refers to an organic molecule having a molecular weight between 50 and 2500 Daltons.

[0055] As used herein, the term "macrocyclic polymer" or "macrocycle" refers to a cyclic polymer or a polymeric cyclic portion of a polymer. Macrocyclic polymers are between 500 Daltons and 7500 Daltons in size. In some examples herein, the macrocyclic polymer is a cyclic peptide or peptide derivative.

[0056] As used herein, the term "binding fragment" refers to a portion of a larger molecule, such as a small molecule, peptide, or antibody, that is expected to directly contact a target protein. Binding fragments can be used for high throughput screening.

[0057] In this disclosure, "binds" or "binding" or "specific binding" and similar terms, when referring to a molecule that "binds" to a protein, such as NALCN, means that the binding affinity is strong enough that the interaction between the members of the binding pair cannot be due to random molecular association (i.e., "non-specific binding"). Thus, the binding is selective or specific.

[0058] As used herein, the term "competitive assay" refers to an assay in which a molecule being tested prevents or inhibits specific binding of a reference molecule to a common target.

[0059] Other definitions are included in the sections below, as appropriate.

[0060] As used herein, "subject" refers to a human. As used herein, "treatment" refers to a clinical intervention, for example, to alleviate at least one symptom associated with a disease or disorder, to slow the progression of one or more symptoms, or to prevent or delay the onset of at least one symptom.

[0061] Isolated human NALCN-FAM155-UNC79-UNC80 complex Some embodiments of the present specification include the formation of an isolated human NALCN-FAM155-UNC79-UNC80 complex in vitro. In some embodiments, the complex is formed by expressing each protein on an isolated vector that can be expressed in a host cell, such as a HEK293 cell, in vitro.

[0062] In some embodiments, at least one of the four proteins includes a label. In some embodiments, the label is used to help bind the protein or protein complex to a matrix such as beads (e.g., streptavidin-coated beads, etc.). Thus, for example, in some embodiments, the label is attached to either or both of the NALCN protein or the FAM155 protein, for example at the C-terminus. In some embodiments, the label can be attached to each of the complex proteins. Examples of such labels are molecules that allow the protein or complex to be attached to another molecule or matrix, such as streptavidin or flag tags, for example, including a peptide segment that is recognized by streptavidin on the surface of the matrix. Another type of label includes labels that allow the detection of the protein or complex by color change, fluorescence, or phosphorescence, such as, for example, a bound small molecule fluorescent label or a co-expressed label such as green fluorescent protein.

[0063] In some embodiments, after expression and formation of the complex in the host cell, the complex can be isolated and resuspended in, for example, lipid bilayers or lipid nanodiscs, or solubilized in detergent. In some embodiments, the complex is resuspended in lipid nanodiodes containing a mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol). In other embodiments, the nanodiscs can contain other lipids, such as POPS (10% palmitoyl-oleoyl-phosphatidylserine) or POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate). In some embodiments, these are mixed in a ratio of 3:1:1. In some embodiments, the complexes may be solubilized in a detergent such as n-dodecyl-BD-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), or glycol-diosgenin (GDN), or a mixture of such detergents. In some embodiments, the complexes resuspended in, for example, lipid bilayers or detergents or lipid nanodiscs may be bound to a matrix such as beads, plates, or chips.

[0064] Similar methods can be used to obtain the UNC79-UNC80 subcomplex or the NALCN-FAM155 subcomplex.

[0065] Exemplary screening methods for identifying human NALCN-FAM155-UNC79-UNC80 complex modulators and binders The isolated human NALCN-FAM155-UNC79-UNC80 complex can be used in a variety of screening experiments, among other potential uses. For example, the complex can be used to screen for modulators of the human NALCN complex. In some embodiments, the method may include: (a) providing in vitro a complex of human NALCN, human FAM155 (family 155 member A with sequence similarity), UNC79 (uncooperative 79) and UNC80 (uncooperative 80) (human NALCN-FAM155-UNC79-UNC80 complex); (b) contacting the complex with a potential modulator of the human NALCN-FAM155-UNC79-UNC80 complex; (c) performing an ion channel assay of the complex in the presence of the potential modulator; and (d) identifying the potential modulator as a modulator of the human NALCN-FAM155-UNC79-UNC80 complex if the activity of the complex in the assay in the presence of the potential modulator is greater or less than the activity of the complex in the assay in the absence of the potential modulator.

[0066] In some embodiments, the activity of the complex in the assay in the presence of the potential modulator is less than the activity of the complex in the assay in the absence of the potential modulator (i.e., the potential modulator reduces the activity of the complex in the assay). For example, in some embodiments, a potential modulator identified in part (c) reduces the activity of the complex in the assay by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some examples, the activity of the complex in the presence of the modulator can be, for example, 1-90%, 10-90%, 1-10%, 1-20%, 25-90%, 50-90%, 25-75%, 40-80%, or 50-75% of the activity of the complex in the absence of the modulator. In some examples, a potential modulator identified in the assay reduces the activity of the complex in the assay at a half-maximal concentration of 10 nM-500 μM, 50 nM-500 μM, 10 nM-50 μM, 100 nM-500 μM, 100 nM-50 μM, 1-500 μM, 1-50 μM, 10-500 μM, or 50-250 μM.

[0067] In some embodiments, the activity of the complex in the assay in the presence of the potential modulator is greater than the activity of the complex in the assay in the absence of the potential modulator (i.e., the potential modulator increases the activity of the complex in the assay). In such examples, a potential modulator identified in the assay increases the activity of the complex in the assay by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 100% (i.e., 2-fold), up to 100%, or up to 3-fold. In some examples, the activity of the complex in the presence of the modulator may be, for example, 10-100%, 10-80%, 20-80%, 25-100%, 25-75%, 50-100%, 2-3-fold, or 100-200% greater than the activity of the complex in the absence of the modulator.

[0068] In any of the above experiments, the activity of the complex may be measured by any of a variety of ion channel assays, including patch clamp or automated patch clamp assays, ion flux assays, or ion or voltage sensitive dye assays. Exemplary assays are described in more detail below.

[0069] In some embodiments, the screening method may further comprise determining the binding affinity of the potential modulator or modulators identified in the assay to the human NALCN-FAM155-UNC79-UNC80 complex and / or one or more of the subcomplexes or individual proteins of NALCN-FAM155 or UNC79-UNC80. For example, ELISA or AlphaLISA or FRET assays may be used to determine the binding affinity. In some embodiments, the potential modulator binds to the human NALCN-FAM155-UNC79-UNC80 complex, or a related subcomplex, or one of the individual proteins of the complex with an EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less.

[0070] In a further embodiment, screening method can be used to identify the molecule that binds to human NALCN-FAM155-UNC79-UNC80 complex.For example, such method can include: (a) providing human NALCN-FAM155-UNC79-UNC80 complex in vitro; (b) contacting the complex with one or more test molecules, and separating bound test molecules from unbound test molecules; and (c) identifying test molecules as the molecule that binds to human NALCN-FAM155-UNC79-UNC80 complex if the test molecules remain bound to the complex after separating bound test molecules from unbound test molecules. In further examples, such methods may include: (a) providing a human NALCN-FAM155-UNC79-UNC80 complex in vitro; (b) contacting the complex with a NALCN-DII-DIII linker peptide or a NALCN-DI-DII linker peptide; and identifying a test molecule as a molecule that binds to the human NALCN-FAM155-UNC79-UNC80 complex if the test molecule competes with the NALCN-DII-DIII linker peptide and / or the NALCN-DI-DII linker peptide for binding to the complex. For example, as described in the following examples, certain NALCN-DII-DIII peptides have been found to modulate and significantly reduce the activity of the complex in an ion channel assay. In some examples, both ion channel assay screening and binding screening may be performed. Also, in some examples, binding assays to the complex may be performed together with binding assays to one of the individual proteins of the NALCN-FAM155 and / or UNC79-UNC80 subcomplexes or complexes. In some embodiments, the NALCN-DII-DIII or NALCN-DI-DII linker peptide may be labeled.

[0071] The screening method herein also includes a method for identifying a molecule that modulates the formation or stability of the complex. An example of such an assay may include a method that includes: (a) providing a human NALCN-FAM155-UNC79-UNC80 complex in vitro; (b) contacting the complex with one or more test molecules and separating bound test molecules from unbound test molecules; and (c) identifying the test molecule as a molecule that modulates the formation or stability of the human NALCN-FAM155-UNC79-UNC80 complex if the degree of complex formation in the presence of the test molecule is increased or decreased compared to the absence of the test molecule. Such an assay may be performed, for example, using FRET or similar labels or AlphaLISA to detect the proximity of various components of the complex in the presence and absence of the test molecule. In some embodiments, such a screening method may further include an additional assay to determine whether the test molecule affects the formation or stability of a subcomplex such as NALCN-FAM155 or UNC79-UNC80.

[0072] In any of the methods herein, one or more components of the complex or potential modulator may be labeled with a label to detect its presence (e.g., via color change, fluorescence, etc.) or its proximity to other complex members (e.g., FRET), or with a label to bind the molecule to a matrix (e.g., streptavidin recognition element or the like). In any of the methods herein, one or more components of the complex or potential modulator may be bound to a matrix, such as a bead, chip, or plate. In any of the above assays, the complex may further include other elements, such as those that adhere to the complex after its expression in vivo in a host cell. In some embodiments, the complex further includes, for example, calmodulin.

[0073] In any of the above methods, the method may be performed using a NALCN-FAM155-UNC79-UNC80 complex, or a related subcomplex that includes one mutant of the protein, such as mutant human NALCN. Exemplary mutant human NALCN polypeptides are described in the Examples and Sequence Listings herein. In some examples, the mutant human NALCN includes substitutions, insertions, or deletions in one or both of the DI-DII or DII-DIII linker elements. These include, for example, deletion of residues, insertion of a flexible element such as a GGGS element (SEQ ID NO: 86) into the linker, or shortening the linker by point mutation in the linker, or shortening part or all of the linker sequence by Na V Human Na, such as 1.4 V or Ca V The present invention may include substitution of a corresponding sequence from another ion channel protein, such as a protein. Examples of human NALCN mutants tested herein include those comprising a sequence selected from SEQ ID NOs: 6-15, 32-52, and 59-63. In other embodiments, a truncated UNC79 or UNC80 polypeptide or a mutant FAM155A molecule may be included in the complex. Examples of human UNC79 mutants include those comprising UNC79 fragments, such as those comprising SEQ ID NOs: 53, 54, 55, and 58, or those comprising a sequence selected from SEQ ID NOs: 64-73. UNC80 fragments, such as those comprising a sequence selected from SEQ ID NOs: 74-85, may also be used. For example, if a test molecule binds to a wild-type complex but does not bind to one or more such mutant complexes, this may provide information about how the test molecule binds to the complex.

[0074] In some embodiments, the screening method is carried out in the presence of NALCN-DII-DIII linker peptide or NALCN-DI-DII linker peptide or another identified modulator of human NALCN-FAM155-UNC79-UNC80 complex. Thus, for example, by including both potential and identified modulators in the assay, it can be determined whether the potential modulator competes with the identified modulator of the activity of the complex. As described in the following examples, it has been found herein that certain linker peptides comprising sequences from the DII-DIII linker of human NALCN reduce or inhibit the function of human NALCN-FAM155-UNC79-UNC80 complex in ion channel assays. Thus, these peptides have been identified as modulators of the complex in the assays described herein. Examples include peptides comprising SEQ ID NOs: 16-18 and 23-24, such as peptides comprising residues 617-740, 659-774 and 617-845 of the DII-DIII linker, optionally with an N-terminal modification such as MBP or palmitoyl, or with an N-terminal methionine residue. In some examples, the peptides may be expressed in a host cell in vivo along with members of a protein complex. In other examples, the peptides may be added separately to the complex in vitro.

[0075] Exemplary Functional Assays In any of the above methods, the ion channel assay can be any suitable assay used to detect the activity of the human NALCN-FAM155-UNC79-UNC80 complex as an ion channel, for example. Examples include, but are not limited to, patch clamp assays, including automated patch clamp assays, ion flux assays, and ion or voltage sensitive dye assays. Exemplary assays are described, for example, in H. Yu et al., “high throughput screening technologies for ion channels,” Acta Pharm. Sinica 37:34-43 (2016), and materials are available from commercial manufacturers. In ion flux assays, for example, sodium 22 ( 22 Na +Radioisotopes such as 1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4) or oxonol derivatives such as FMP may be used to track the cellular influx and efflux of sodium or other ions. Another type of assay is the voltage-sensitive or ion-sensitive dye assay. In the voltage-sensitive dye assay, the voltage change across a membrane containing an ion channel protein is measured using fluorescence resonance energy transfer (FRET) using a dye such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4) or an oxonol derivative such as FMP. In some examples, the FRET dye may be localized or tethered to the membrane. The ion-sensitive dye assay may use a dye that shows a difference in signal depending on the ion concentration. One example is the sodium indicator dye SBFI. In another embodiment, a patch clamp assay may be used for the screening method. In some embodiments, an automated patch clamp assay may be used. Exemplary platforms and instruments for performing patch clamp assays are sold by several manufacturers. Examples of platform assays include IonWorks™ platform assays, PatchXpress™ and IonFlux™ (Molecular Devices), Qpatch™ HT / HTX (Sophion), and Patchliner™ and SynchroPatch™ (Nanion Technologies).

[0076] In some embodiments, two-electrode voltage clamp assay is carried out in Xenopus oocytes. In this assay, oocytes are injected with a 1:1:1:1 mixture of mRNA or cDNA (or the like) of each member of the complex to express the complex proteins. In some embodiments, the identified modulator of the complex can be further expressed, for example, a soluble linker peptide, for example, DI-DII or DII-DIII linker or a part thereof. Then, two-electrode voltage clamp assay can be carried out, for example, as described in the following examples.

[0077] In some embodiments, patch clamp assay can be performed in HEK-293T cells, for example.In some embodiments, complex is formed from, for example, NALCN-eGFP-2xFLAG labeled NALCN, UNC79, UNC80 and FAM155A transfected cDNA ratio of 2:1:1:1 (or similar).More details are provided in the examples.

[0078] In any of the above methods, the potential modulator identified in the method modulates the activity of human NALCN-FAM155-UNC79-UNC80 complex, for example, in the presence of the modulator, the complex has lower or higher activity than without the modulator. In some examples, the potential modulator reduces the activity of the complex. In some examples, the activity can be reduced by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90%.

[0079] In any of the above methods, the ion channel assay can also be performed in the presence of both a potential modulator and an identified modulator of the human NALCN-FAM155-UNC79-UNC80 complex. For example, in such an example, it can be determined whether the identified modulator competes with the potential modulator to change the activity of the protein. Examples include peptides comprising the amino acid sequences of SEQ ID NOs: 16-18 and 23-24.

[0080] Any of the above methods may further comprise determining the binding affinity of the potential modulator identified in the assay for the human NALCN-FAM155-UNC79-UNC80 complex or its subcomplexes or individual proteins and variants thereof. For example, such assays may help understand the mechanism by which molecules regulate the activity of the complex. For example, an ELISA assay may be used to determine the binding affinity using a lipid-stabilized form of the human NALCN-FAM155-UNC79-UNC80 complex on a matrix, such as a solid surface, such as a bead, plate, etc. The beads may have any shape, such as flakes or chips, spheres, pellets, etc. In some embodiments, such beads are streptavidin-coated beads, avidin-coated beads, or deglycosylated avidin-coated beads. In some embodiments, such beads are magnetic beads. In some examples, the potential modulator binds to the human NALCN-FAM155-UNC79-UNC80 complex or a subcomplex or individual protein thereof with an EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less.

[0081] In some embodiments, further experiments can be carried out on the molecules selected in the above screening, for example, to determine other biological activities of the molecules.For example, further assays can be used to determine whether the molecule also binds to other ion channel proteins or to the mutants of human NALCN-FAM155-UNC79-UNC80 complex protein, thus determining the specificity of the molecule as ion channel modulator or binder.

[0082] Test molecules for screening methods In any of the above methods, the potential modulator (i.e., test molecule) can be a peptide or macrocyclic polymer or an antibody.For example, in some examples, the potential modulator is a small molecule.The present disclosure also relates to a modulator of human NALCN or human NALCN-FAM155-UNC79-UNC80 complex identified by the methods described herein, which can be a peptide, a macrocyclic polymer, a small molecule or an antibody.

[0083] In some embodiments, the potential modulator molecules being tested are peptides. In some embodiments, the peptides are 3-40 mers, 3-20 mers, 4-16 mers, 4-14 mers, or 6-14 mers, such as 3-mers, 4-mers, 5-mers, 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, 25-mers, 26-mers, 27-mers, 28-mers, 29-mers, 30-mers, 31-mers, 32-mers, 33-mers, 34-mers, 35-mers, 36-mers, 37-mers, 38-mers, 39-mers, or 40-mers.

[0084] In some embodiments, the peptide is a macrocyclic polymer. In some embodiments, the macrocyclic polymer is a 3-40 mer, 3-20 mer, 4-16 mer, 4-14 mer, or 6-14 mer, such as a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer macrocyclic compound.

[0085] In some embodiments, the molecule tested in the screening herein is a small molecule. In some embodiments, the molecule tested is an antibody, which may include any full-length antibody of IgG, IgM, IgA, IgD, and IgE, as well as antigen-binding fragments of antibodies such as Fv, Fab', (Fab')2, scFv, nanobodies, single-chain antibodies, bispecific or multispecific antibodies.

[0086] In some embodiments, the molecule being tested is a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody (eg, an antigen-binding fragment).

[0087] In some embodiments, for example, a test molecule identified as a modulator of the NALCN-FAM155-UNC79-UNC80 complex can be used in a method for treating a disorder associated with dysfunction of the NALCN-FAM155-UNC79-UNC80 complex. In some embodiments, a subject having dysfunction of the NALCN-FAM155-UNC79-UNC80 complex can be treated, for example, for channelopathies, neurodevelopmental disorders, circadian rhythm disorders, or pain. Alternatively, in other embodiments, a molecule identified herein can be useful, for example, for treating channelopathies, neurodevelopmental disorders, circadian rhythm disorders, or pain in a subject not having dysfunction of the NALCN-FAM155-UNC79-UNC80 complex. For example, a molecule identified herein can, in some embodiments, modulate the function of the complex in a subject. Channelopathies as defined herein include diseases or disorders characterized by dysfunction of one of the members of the NALCN complex or various potassium or calcium channel proteins.

[0088] molecular complex In some embodiments, the disclosure includes molecular complexes comprising human NALCN-FAM155-UNC79-UNC80, human NALCN-FAM155, human UNC79-UNC80, or human NALCN as described herein conjugated to a molecule such as a peptide, a small molecule, an antibody, or a potential modulator molecule such as a binding fragment of a peptide, small molecule, or antibody.

[0089] In some embodiments, the molecule is a peptide. In some embodiments, the peptide is a 3-40 mer, a 3-20 mer, a 4-16 mer, a 4-14 mer, or a 6-14 mer, such as a 3 mer, a 4 mer, a 5 mer, a 6 mer, a 7 mer, a 8 mer, a 9 mer, a 10 mer, a 11 mer, a 12 mer, a 13 mer, a 14 mer, a 15 mer, a 16 mer, a 17 mer, a 18 mer, a 19 mer, a 20 mer, a 21 mer, a 22 mer, a 23 mer, a 24 mer, a 25 mer, a 26 mer, a 27 mer, a 28 mer, a 29 mer, a 30 mer, a 31 mer, a 32 mer, a 33 mer, a 34 mer, a 35 mer, a 36 mer, a 37 mer, a 38 mer, a 39 mer, or a 40 mer.

[0090] In some embodiments, the peptide is a macrocyclic polymer. In some embodiments, the macrocyclic polymer is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, such as a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer macrocyclic compound.

[0091] In some embodiments, the molecule in the complex is a small molecule. In some embodiments, the molecule is an antibody, which may include any full-length antibody, such as IgG, IgM, IgA, IgD, and IgE, as well as antigen-binding fragments of antibodies, such as Fv, Fab', (Fab')2, scFv, nanobodies, single-chain antibodies, bispecific or multispecific antibodies.

[0092] In some embodiments, the molecule is a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody (eg, an antigen-binding fragment).

[0093] kit The present disclosure also includes a kit that includes a reagent related to the screening method herein. In some examples, the kit includes one or more of the human NALCN, FAM155, UNC79 and UNC80 proteins described herein, or an isolated nucleic acid, vector or host cell for use in expressing a protein for purification of a complex of the four proteins. In some examples, the kit may include one or more mutants of a protein, such as a protein that is linked to a label for detection or binding a matrix. In some examples, the kit includes a reagent used in the screening method herein, with or without a protein complex component. In some examples, the kit includes two or more NALCN, FAM155, UNC79 or UNC80 proteins, such as a mutant protein and a wild-type protein.

[0094] In some embodiments, the kits herein may include one or more reagents for performing an ion channel assay. In some examples, the kits may include a specific identified modulator of the human NALCN-FAM155-UNC79-UNC80 complex, for example as a positive control. The kits herein may also include a negative control identified as not modulating the human NALCN-FAM155-UNC79-UNC80 complex. The kits may also include one or more of NALCN, FAM155, UNC79 or UNC80, or complexes thereof, bound to a matrix particle, such as a bead, or another type of matrix, such as a plate. The beads may have any shape, such as a flake or chip, a sphere, a pellet, etc. In some embodiments, such beads are streptavidin-coated beads, avidin-coated beads, or deglycosylated avidin-coated beads. In some embodiments, such beads are magnetic beads. The protein complexes may or may not be pre-bound to a matrix. In some embodiments, reagents are included to facilitate binding of the protein to the matrix, such as via biotin-streptavidin or similar systems.

[0095] In some embodiments, the kits may include some or all of the reagents relevant to the screening methods herein, such as the reagents necessary to perform the ion channel assays described in the methods herein. In some instances, one or more control reagents, such as modulators of the protein, may also be included.

[0096] In some embodiments, the kit may include a test molecule or library of test molecules, such as a peptide, a small molecule, and / or an antibody. In some embodiments, the peptide in the kit may be a macrocyclic polymer. In some embodiments, the kit may include test molecules that are peptides, small molecules, or binding fragments of antibodies.

[0097] In some embodiments, the kit may also include instructions for use. EXAMPLES

[0098] Example 1. General Method A. Expression and purification of the NALCN-FAM155A-UNC79-UNC80-CaM complex Optimized coding DNA for human NALCN, FAM155A, UNC80 and UNC79 were cloned into pRK vector behind the cytomegalovirus (CMV) promoter, respectively. A twin-Strep-2xFlag tag was added to the N-terminus of NALCN, and a 2xFlag tag was added to the C-terminus of FAM155A, UNC80 and UNC79. Expi293 cells in suspension were cultured in SMM 293T-I medium at 37°C under 5% CO2 until a cell density of 4 × 10 6 When cells / ml were reached, DNA was transfected with polyethyleneimine (PEI) at a ratio of 1:1:1:1. Transfected cells were cultured for 48 hours and then harvested.

[0099] Sixty grams of the harvested cell pellet was resuspended in 300 mL of 25 mM HEPES pH 7.5, 200 mM NaCl, 1 μg / mL benzonase, 1 mM PMSF and Roche protease inhibitor tablets. Cells were lysed by dounce homogenization, followed by solubilization of NALCN complexes by addition of 2% (w / v) glycol-diosgenin GDN supplemented with 0.1% (w / v) cholesteryl hemisuccinate and 0.2 mg / mL porcine brain polar lipid extract (Avanti) for 2 hours at 4°C under gentle agitation. Insoluble debris was stirred at 125,000 x g max The solubilized proteins were pelleted by ultracentrifugation at 4°C for 1 h, and the supernatant containing the solubilized proteins was collected and affinity purified by batch binding to 5 mL of M2-agarose FLAG resin (Sigma) for 1 h at 4°C. Unbound proteins were washed with 6 column volumes (CV) of purification buffer (6 CV 25 mM HEPES pH 7.5, 200 mM NaCl, 0.04% (w / v) GDN followed by 10 CV of purification buffer supplemented with 500 mM NaCl, and 10 CV of buffer supplemented with 5 mM ATP and 10 mM MgCl2). NALCN was eluted with 5 CV of purification buffer supplemented with 300 μg / mL of FLAG peptide (Sigma). The eluate was collected and applied to 3 mL of Strep-Tactin XT high affinity resin (IBA) and allowed to bind in batch for 3 h. Unbound proteins were washed with 10 CV of purification buffer and eluted with 5 CV of purification buffer supplemented with 50 mM biotin. The NALCN complex was then concentrated to 4 mg / mL with an Amicon® Ultra centrifugal filter device (100 kDa MWCO) concentrator.

[0100] B. Reconstitution of NALCN channelosomes into nanodiscs A 200-fold molar excess of a 3:1:1 lipid mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) (presolubilized by sonication at 10 mg / mL in buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 1% CHAPS) was added to an aliquot of 0.35 nmol of NALCN in a 2 mL tube and incubated for 30 min at 4° C. A 4-fold molar excess of the membrane scaffold protein MSP1E3D1 (Sigma) was then added and incubated for an additional 30 min at 4° C. before diluting to 1.5 mL with purification buffer. BioBeads were added to a concentration of 0.25 mg / mL and the sample was incubated overnight at 4° C. with rotation. The reactions were then combined and the Bio-Beads were removed before the reaction was concentrated to 100 μL. The concentrated reaction was applied to a Superose® 6 3.2 / 300 column pre-equilibrated with Gel Filtration Buffer (25 mM HEPES pH 7.5, 200 mM NaCl). Peak fractions were pooled and concentrated to 2.4 mg / mL.

[0101] C. Cryo-EM Sample Preparation and Data Acquisition NALNC-FAM155A-UNC79-UNC80-CaM complex grids were prepared in the following manner: Holly carbon grids (Ultrafoil™ 25 nm Au R 0.6 / 1 300 mesh; Quantum Foil) were glow discharged for 30 seconds using a Solarus® plasma cleaner (Gatan). NALNC-FAM155A-UNC79-UNC80-CaM complexes were gently cross-linked with 0.05% EM grade glutaraldehyde for 10 minutes at room temperature. Cross-linking was quenched with 9 mM Tris pH 7.5 and 3 μL of sample was applied to the grid. Grids were blotted in a Leica Microsystems automated plunge freezer (EM GP2 Leica Microsystems) using a blotting time of 3.5 seconds at 100% humidity and plunger frozen in liquid ethane cooled with liquid nitrogen. Serial EM on Titan Krios™ operated at 300 keV with a BioQuantum™ energy filter equipped with a K3™ Summit direct electron detector camera (Gatan) 1 Movie stacks were collected from two grids using a 20 eV energy slit at 105,000x magnification, corresponding to 0.838 Å / pixel. Each image stack had a resolution of approximately 60 e / Å. 2 It contains 60 frames recorded every 0.05 s for a cumulative dose of 1000 nm and a total exposure time of 3 s. Images were recorded at a set defocus range of 0.5-1.5 µm.

[0102] D. Cryo-EM Data Processing RELION(trademark) 34 and cisTEM(trademark) 35 The cryo-EM data were processed using a combination of software packages: MotionCor2 from Relion; 36 We compensated the 15,080 videos for frame motion using our implementation of CTFFIND-4™. 37Contrast transfer function (CTF) parameters were fitted using a bandpass of 30–4.5 Å of the spectrum using WARP™. Images were filtered based on the detected fit resolution better than 8 Å. A retrained deep learning-based selection algorithm was used to filter the images. 381,778,009 particles were screened using . To obtain the first 3D reconstruction, particles were rigorously screened in three rounds of reference-free 2D classification using 100-200 classes for each classification to select the best aligned particles. 446,099 particles were then subjected to 3D classification in RELION™. The resulting 3D classes served as 20 Å LPF criteria for a second 3D classification performed on 233,310 selected particles (two of the four classes). To improve the quality of the 3D reconstruction, the 3D map and 76,673 selected particles from a single 3D class were exported from RELION™ and imported into cisTEM™ for iterative rounds of maskless automatic refinement and manual refinement with successively adjusted masks by applying LPF outside the mask (filter resolution 20 Å, outer weight 0.8). The resulting 3D reconstruction was subjected as a reference map for an additional round of RELION™ 3D classification using 446,099 particles after 2D classification, followed by cisTEM™ automatic and manual refinement. The iterations between RELION™ 3D classification and cisTEM™ refinement were repeated until the resolution and quality of the resulting EM reconstructions converged. The 3D reconstruction was then subjected as a 3D reference for RELION™ 3D classification with an extensive selection of 1,718,298 particles, after a single round of reference-free 2D classification with 300 classes to separate only NALCN complex particles from debris and other false positives. 1,007,024 particles from four of the six selected classes were exported from RELION™ and imported into cisTEM™ for iterative rounds of maskless automatic refinement and manual refinement with a mask by applying an LPF (filter resolution 20 Å, outer weight 0.8) outside the mask. Refinement was repeated until the resolution and quality of the resulting EM reconstructions converged.After 3D classification and multi-model manual refinement in cisTEM™ (separation of the 6 classes), 365,512 particles (≈38%) from a single class were extracted and subjected to unmasked cisTEM™ automatic refinement, followed by manual refinement with a mask by applying LPF (filter resolution 20 Å, outer weight 0.8) outside the mask and a score threshold of 0.25. The 3D reconstruction converged to a map resolution of 2.8 Å (FSC=0.143, as determined in cisTEM™). For model building and drawing, the maps were sharpened in cisTEM™ with the following parameters: flattening from 8 Å resolution, -90 Å from the origin of reciprocal space. 2 Application of pre-cutoff B-factor, figure of merit filter 39 The local resolution is achieved by applying the blocres algorithm. 40 The results were determined in a cisTEM™ using an in-house reimplementation of the .

[0103] E. Model Building and Structural Analysis The structures of NALCN-FAM155A (PDB:6XIW) and CaM (PDB:4DCK) were used as templates to model the NALCN complex and rigid-body fitted to the cryo-EM map. The structures of UNC79 and UNC80 and the NALCNDI-DII and DII-DIII linker regions were de novo built into the map. After extensive (re)building and manual refinement, the phenix.real_space_refinement 41 Global structural differences between the initial model and the map were corrected using multiple real-space refinements using the tool, ISOLDE™, through iterative rounds of model building and real-space refinement in Phoenix. 44 Using Coot 42 and UCSF ChimeraX 43 The model was further manually tuned using MolProbity scoring. 46 phenix.validation_cryoem 45The results were verified using PyMOL (trademark) (The PyMOL Molecular Graphics System, version 2.07 Schrodinger, LLC), UCSF Chimera 47 or UCSF ChimeraX 43 Diagram created using DALI Server 32 3D homology structure analysis was performed using JalView™ 49 Clustal Omega™ 48 Sequences were aligned using ESPrimpt 3.0™ 50 was used as an example and then manually adjusted based on insights from the NALCN-FAM155A-UNC79-UNC80-CaM model.

[0104] F.2 Electrode voltage clamp electrophysiology testing Xenopus laevis oocytes were prepared as previously described. 10 Healthy appearing stage V-VI oocytes were isolated and injected with 30-40 ng of RNA in a volume of 32-41 nl using a Nanoliter 2010 injector (World Precision Instruments). V1.4 chimera), UNC-79, UNC-80 and FAM155A RNAs were mixed in a 1:1:1:1 ratio. When expressing NALCN, UNC79, UNC80, FAM155A with isolated linkers, the RNAs were mixed in a 1:1:1:1:1 ratio. For the control combination (i.e., NALCN+UNC79+UNC80+FAM155A without linkers), the concentration of each RNA was kept constant by adding an equal volume of nuclease-free water. Injected cells were incubated at 18°C ​​and 140 rpm in ND96 storage solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, 2.5 mM pyruvate, 0.5 mM theophylline; pH adjusted to 7.4 with NaOH) supplemented with 50 μg / mL gentamicin and tetracycline. Four to five days after RNA injection, two-electrode voltage-clamp measurements were performed on oocytes continuously perfused in ND96 recording solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM Hepes (pH 7.4) with NaOH) at room temperature using a Warner OC-725C Oocyte Clamp amplifier (Warner Instrument Corp., USA). Data were acquired using pCLAMP™ 10 software (Molecular Devices) and a Digidata® 1550 digitizer (Molecular devices) and sampled at 10 kHz. Electrical powerline interference was filtered with a Hum Bug 50 / 60 Hz Noise Eliminator (Quest Scientific). Recording microelectrodes with resistances of approximately 0.2 M to 1.0 MΩ were pulled from borosilicate glass capillaries (Harvard Apparatus) using a P-1000 Flaming / Brown Micropipette Puller System (Sutter Instrument) and filled with 3 M KCl.

[0105] G. Patch Clamp Electrophysiology Testing HEK-293T cells were maintained as previously described (M. Kschonsak et al., Nature 587:313-318 (2020)). Cells between passages 6 and 20 were used for experiments and tested for mycoplasma (Eurofins Genomics). For patch clamp experiments, cells that reached 40–60% confluence in 35 mm cell culture dishes were transiently transfected with the constructs of interest using LipoD293 ver.II (tebu-bio) 20–24 h prior to recording. A total of 2.5 μg (for LipoD293) of cDNA was used. NALCN-eGFP-2×FLAG, UNC79, UNC80 and FAM155A cDNAs were mixed in a mass ratio of 2:1:1:1.

[0106] On the day of the experiment, transfected HEK-293T cells were seeded on glass coverslips coated with poly-L-lysine at least 3 h prior to recording. Cells were voltage clamped in whole-cell configuration at room temperature using an Axopatch™ 200B amplifier (Molecular Devices). Data were acquired at 10 kHz using pCLAMP™ 10 software (Molecular Devices) and an Axon™ Digidata® 1550A digitizer (Molecular Devices). Patch pipettes were extracted from Kwik-Fil 1.5 / 1.12 [outer diameter (OD) / inner diameter (ID) in millimeters] borosilicate glass capillaries (World Precision Instruments) and flame-polished to resistances of approximately 3.0 MΩ–5.5 MΩ. Extracellular solutions were rapidly exchanged using a custom-made glass perfusion tool with four adjacent barrels (OD / ID 0.45 / 1.60, in millimeters; CM Scientific) controlled by an MXPZT-300R solution switcher (Siskiyo).

[0107] Symmetric Na +For the conditions, (1) the extracellular solution contained 150 mM NaCl with NaOH, 10 mM HEPES, and 30 mM D-(+)-glucose (pH 7.4) and was approximately 325 mOsm / L, and (2) the intracellular solution contained 136 mM NaCl with NaOH, 10 mM NaF, 5 mM EGTA, 10 mM HEPES, and 2 mM Na2ATP (pH 7.2) and was approximately 309 mOsm / L. For conditions more similar to physiological conditions, (1) the extracellular solution contained 150 mM NaCl with NaOH, 5 mM KCl, 0.5 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 13 mM D-(+)-glucose (pH 7.4) at approximately 320 mOsm / L, and (2) the intracellular solution contained 140 mM CsCl with CsOH, 10 mM CsF, 5 mM EGTA, 10 mM HEPES, and 2 mM Na2ATP (pH 7.2) at approximately 304 mOsm / L. To prevent nonspecific leakage from affecting the accuracy of our results, we routinely checked for loose seals by exposing the cells to an extracellular solution of NMDG only before and / or after the experiment. Cells that exhibited a steady-state inward current greater than 10 pA at -80 mV in the absence of permeant ions were discarded.

[0108] Example 2. UNC79 / UNC80-NALCN-linker-based fluorescence-based polarization assay for screening channel modulators Recombinant UNC79-UNC80 complex is expressed and purified as described in Example 1A. In particular, full-length human UNC79 is cloned into a pRK (or analog) vector behind a CMV promoter (or analog) with a carboxy-terminal Flag tag (or analog). Full-length human UNC80 is cloned as UNC80, but with a carboxy-terminal streptavidin-tag (or similar). Both plasmids are co-transfected into Expi293 cells using standard protocols and expressed for 48 hours at 37°C. Cell pellets are harvested and dissolved in standard non-denaturing buffer supplemented with protease inhibitors. UNC79-UNC80 complex is purified using standard protocols for anti-Flag affinity purification followed by anti-streptavidin tag affinity purification. The harvested UNC79-Flag-tag:UNC80-streptavidin-tag complex is further purified on a Superose® 6 column and the peak monodisperse fraction is harvested. Save the purified protein fractions. Measure and adjust the concentration of the purified protein. Divide the purified protein into aliquots and freeze until further use.

[0109] Synthetic peptides containing the target intracellular loop regions from human NALCN are synthesized using standard peptide chemistry methods with appropriate fluorescent dyes (e.g., Alexa647, Alexa488, TAMRA and Cy5) conjugated to either the N- or C-terminus of the peptide, either directly or via a PEG6 linker or the like. These synthetic peptides contain residues from the DI-DII loop (Gln349-Ala363; SEQ ID NO:19) and / or the DII-DIII loop (Ser617-Arg845; SEQ ID NO:18) of human NALCN. Shorter synthetic peptides from any of these intracellular regions (e.g., 638-670, 794-828 and / or 829-835; SEQ ID NOs:20, 21 and 22, respectively) may also be considered for conjugation to appropriate fluorescent dyes. Unlabeled versions of these same peptides and random scrabble peptides are also generated as assay reagents.

[0110] Purified UNC79:UNC80 complexes and synthetic fluorescent NALCN-based peptides are used to develop and optimize the assay using standard fluorescence-based plate readers in 384-well or 1586-well formats. Fluorescent NALCN-based peptides are used to measure binding of the NALCN linker peptide to the UNC79-UNC80 complex. Unlabeled NALCN peptides or scrambled peptides are used along with the fluorescent NALCN peptide. The unlabeled peptides compete with the fluorescent peptide for binding to the UNC79-UNC80 complex and may even displace the fluorescent peptide bound to the UNC79-UNC80 complex. The scrambled peptide has no effect. Example assay parameters include protein and peptide concentrations, type of dye and dye conjugation site used, buffer conditions, and assay temperature are optimized to maximize the signal and sensitivity and robustness of the assay.

[0111] Once appropriate assay parameters have been determined, the UNC79-UNC80 complex and fluorescent NALCN-based peptides are screened against a diverse chemical library to identify molecules that can compete with (and displace) the peptides binding to the UNC79-UNC80 complex. Molecules identified from the screen are further characterized by dose-response assays and then tested for modulation of NALCN channel activity using standard electrophysiological assays.

[0112] Example 3. Characterization of recombinant NALCN channelosomes Previous efforts to purify the intact NALCN-FAM155A-UNC79-UNC80 complex only isolated the NALCN-FAM155A subcomplex. 24-26 We note that fusion of the carboxy-terminal tagged green fluorescent protein (GFP-Flag) to the NALCN subunits has no apparent effect on their functional properties (Figure 1A), and thus we have demonstrated that stable NALCN GFP-This allowed the isolation of the FAM155A-UNC79-UNC80 complex (Figure 5A-C).

[0113] NALCN GFP The FAM155A-UNC79-UNC80-CAM complex was reconstituted into lipid nanodiscs and then purified using CA 2+ The samples were vitrified for cryo-electron microscopy (CRYO-EM) examination without replenishment (Figures 5C-D). Our 3D reconstructions, extending to a resolution of approximately 3.1 Å, revealed all four components of the NALCN-FAM155A-UNC79-UNC80 channelosome, as well as CaM (Figures 1B-F and 5E-H; Table 1). Due to the unique properties of the sample (Figures 5F and 6A-B), we were not able to assign complete atomic models to all regions of the map. [Table 1]

[0114] Example 4. Overall structure of the NALCN channelosome Although unprecedented among ion channels, the structure of the NALCN channelosome complex is reminiscent of a bicycle when viewed perpendicular to the membrane (Figure 1B-C). The characteristic structure of the complex is provided by UNC79 and UNC80, which form a giant, tangled intracellular assembly that hangs below the voltage sensor domain 1 (VSD1), VSD2, and VSD3 on NALCN (Figure 1B-F). Below VSD4, the C-terminal domain (CTD) of NALCN is encompassed by CaM, which is itself docked to UNC80 near the midpoint of the intracellular assembly (Figure 1D-E).

[0115] The overall conformation of the NALCN-FAM155A pore-forming subcomplex is largely unchanged within the channelosome compared to the unliganded subcomplex ( Figure 7A ). 24-26 FAM155A remains structurally unchanged (Fig. 7B), supporting the conclusion that it functions primarily to stabilize the NALCN subunit while shielding the selectivity filter from neurotoxin attack. 25In addition, the two predicted transmembrane helices of FAM155A remain unresolved (Figure 5F), indicating that they act as simple membrane anchors for the extracellular domain. 10 Importantly, in the presence of UNC79, UNC80 and CaM, NALCN pore-forming subunits displayed extensive ordering of the intracellular linker region (Figures 1B and 7A). As we discuss below, these key structural features have important consequences for NALCN channelosome assembly and gating.

[0116] Example 5. UNC79 and UNC80 are atypical HEAT repeat proteins Contrary to all predictions, UNC79 and UNC80 are well-folded globular proteins (Fig. 1B-F, Fig. 2A-P and Fig. 8A-B). UNC79 and UNC80 form a tight head-to-tail superhelical assembly reminiscent of an infinite sign that extends about 300 Å beneath the membrane bilayer (Fig. 1C and Fig. 2A). No transmembrane helices or membrane interactions were observed (Fig. 5F and Fig. 8A-B), indicating that UNC79 and UNC80 can tightly associate with NALCN pore-forming subunits through intracellular contacts (Fig. 1C-F). There is no known N-terminal domain between UNC79 and UNC80. UNC79 - CUNC80 (NC) interfaces, crossover interfaces, and CUNC79 -N UNC80 Three large interaction interfaces at the (C-N) interface are formed (Figure 2A-D).

[0117] UNC79 and UNC80 are readily assigned to the HEAT repeat superfamily fold (Figure 2A), defined by a repeated motif of two linked α-helical, with some HEAT repeats being irregular and bearing more three α-helical armadillo repeat-like features (Figure 2A, Tables 2 and 3). [Table 2] [Table 3]

[0118] UNC79 (2,635 residues) contains 70 modeled α-helices representing 32 HEAT repeats, whereas UNC80 (3,258 residues) contains 72 modeled α-helices representing 31 HEAT repeats (Figure 2A and Figure 8A-B). The folded and highly intertwined structures of UNC79 and UNC80 may ultimately explain their unusually high sequence conservation among mammals (Figures 9-10). Compared to UNC80, UNC79 has less deviation in the length of α-helices throughout its structure and is more regular in shape and dimensions, rationalizing why this smaller protein contains more HEAT repeats (Figure 2A and Figure 2F-J). UNC80 has many exceptional HEAT repeat units with α-helices extending up to 60 residues in length and has an elaborate structure with regular and irregular substructures, including the incorporation of ubiquitin (Ub)-like folds near the NALCN-CTD and CaM interaction sites (Figures 2A and 2F-L). Both UNC79 and UNC80 also contain numerous unresolved loops along their respective scaffolds, including an irregular region of approximately 600 residues spanning 1437-2014 in UNC79 (Figures 8A-B). Overall, UNC80 appears thicker and less uniform when compared to UNC79, but structural overlay reveals that a highly similar structure is shared between UNC79 and UNC80, despite no discernible sequence identity (Figures 2F-J).

[0119] Because HEAT repeat proteins are ubiquitous, it is surprising that UNC79 and UNC80 are the first examples incorporated into any ion channel (Tables 2 and 3). DALI search of the Protein Data Bank 32returned importin-α3 as the top hit, which can be superimposed end-to-end three times over the length of UC79 and UC80 (Figure 2I-J). Beyond their rough relationship to importin-α3, UNC79 and UNC80 differ substantially from canonical HEAT repeat proteins with respect to their α-solenoid structural pitch (Figure 2O-P). These striking structural differences may explain the lack of strong sequence identity to other HEAT repeat proteins (Tables 2 and 3) and suggest that the UNC79 and UNC80 subunits have evolved highly specialized roles in the assembly and function of the NALCN channelosome complex.

[0120] Example 6. UNC79 and UNC80 form integrated structural modules Three extensive and conserved interaction interfaces are observed between UNC79 and UNC80, giving the impression that these subunits form a stable subcomplex (Figure 2A-H). Both UNC79 and UNC80 are S-shaped and dock with each other in a complementary manner along an apparent degenerate pseudo-2-fold axis (Figure 2A and 2I-J). Recognizable structural features are found at each end of the complex, with the NC domain stabilized by a C-terminal helix contributed by UNC80 and the CN domain similarly capped by a C-terminal helix contributed by UNC79 (Figure 2A-C).

[0121] A crossover interface at the midpoint of the complex explains why both UNC79 and UNC80 are essential for NALCN channelosome assembly and function (Figures 2B and 2D). This conserved interface reveals a high degree of surface and electrostatic complementarity (Figures 2F-H) and spans approximately 3,000 A. 2The UNC79-UNC80 buries an impressive solvent-accessible surface area of ​​100 nm. Indeed, a complex mixture of polar and hydrophobic features is conserved along all UNC79-UNC80 interfaces, determined by the unique size and geometry of the assembly (Figures 2G and 2H). Thus, UNC79 cannot recapitulate UNC80 interactions, or vice versa, and so channelosomes do not form in the absence of either subunit. Although it is unclear whether UNC79 or UNC80 can exist alone in cells, the exposure of such a large hydrophobic surface could be expected to affect protein expression, folding, or stability of the individual subunits. In support of this notion, it has been shown that all intracellular UNC79 and UNC80 are NALCN-associated. 12 and protein expression levels and localization also correlate 3、17、30 .

[0122] Example 7. UNC79-UNC80 subcomplex affects NALCN activity To examine the UNC79-UNC80 subcomplex, we first systematically co-expressed overlapping ∼500 residue fragments from each subunit in the context of the NALCN channelosome in Xenopus oocytes (Figures 11A-11F). No fragments gave rise to dominant gating phenotypes expected as alterations in inward or outward currents, highlighting the fidelity with which full-length UNC79 and UNC80 subunits coassemble (Figures 11A-11F). We then evaluated constructs with truncations or internal deletions and found that NALCN channelosome function tolerated deletion of either the C-terminal region of UNC79 (Δ2401-2635) or the N-terminal region of UNC80 (Δ1-733), respectively (Figures 2E, 2Q-2T, and 11G-11I). 10 This indicates that part of the CN interface is not necessarily required (Fig. 2A). In contrast, NALCN channelosome function is consistent with previous suggestions that UNC79 forms an essential interaction with UNC80. 12Consistent with this, UNC79 is sensitive to deletions at the NC interface (Figures 2A, 2E, 2Q-T, 11G-I). We then split UNC79 at three positions and co-expressed the complementary fragments with intact NALCN, FAM155A and UNC80. NALCN channelosome function tolerated fragmentation of UNC79 at Leu467 or Cys800, but truncation of UNC79 at Lys1400 resulted in loss of function (Figures 2E, 2Q-T), indicating that disruption of the crossover interface is not tolerated (Figure 2A).

[0123] In humans, biallelic mutations in UNC80 cause autosomal recessive disorders in which the pathogenic variant may arise as a nonsense mutation. 12、28、29 Three nonsense UNC80 mutations (Arg51X, Arg174X, Arg2706X) failed to elicit currents when co-expressed with NALCN, FAM155A and UNC79 in oocytes (Figure 2E and Figures S11J-S11L). Structurally, all of these mutations are predicted to be deleterious to UNC80 folding or assembly in the NALCN channelosome, similar to previously evaluated UNC80 nonsense mutations (Figures 2A and S11E). 12、28,29 Two biallelic UNC80 missense mutations (Val189Met and Pro1700Ser) were evaluated and showed a slight reduction in current amplitude, indicating partial loss of NALCN channelosome function (Figure S11M-N). The Val189Met mutation maps within the hydrophobic core of UNC80 and may be destabilizing, whereas the Pro1700Ser mutation is near the UNC79-NALCN interface that appears to be important for channelosome function, as described below (Figure S11E). Furthermore, the biallelic UNC80 mutant Q341H-P342S, which was recently shown to disrupt NALCN channelosome function, 12would be expected to damage and destabilize the CN interface within the UNC79-UNC80 subcomplex (Figure 11E). Overall, we conclude that UNC79 and UNC80 form an essential and interdependent subcomplex, and disruptive mutations may adversely affect NALCN channelosome assembly, trafficking or function.

[0124] Example 8. NALCN linker contacts are essential for the UNC79-UNC80 subcomplex NALCN subunits use their intracellular DI-DII and DII-DIII linkers to interact on either side of the UNC79-UNC80 crossover interface, which is structurally reminiscent of the strings on a marionette puppet (Figure 3A-C). Part of the DI-DII linker engages the membrane-proximal surface of UNC79, while two distinct regions from the DII-DIII linker form elaborate clamp-like interactions on UNC80 (Figure 3A-G). These anchoring interactions are different from those previously seen in other ion channel-auxiliary subunit complexes, and are consistent with previous work in the 2000A. 2 The inventors have demonstrated that the DI-DII, DII-DIII and DIII-DIV linkers and Na V We systematically substituted the N- or C-terminus of 1.4 with NALCN and found an absolute requirement for DI-DII and DII-DIII linkers to support NALCN channelosome activity (Figures S12A-G).

[0125] The NALCNDI-DII linker is approximately 40 residues long (Met335-Ala375) with only a resolved hairpin loop that inserts Phe351, Trp359, and Leu361 into a conserved hydrophobic cleft between HEAT repeats (HR)-64 and HR-65 of UNC79 (Figure 3A, 3C, 6A). Deletion of this hairpin loop (ΔGln349-Ala363) abolished NALCN channelosome function, whereas deletion of single residues (ΔPhe351, ΔTrp359, and ΔLeu361) established the essential role of Trp395 in anchoring this critical NALCN-linker-UNC79 interface (Figure 3I-J and Figures S13A-B).

[0126] The NAL CNDII-DIII linker is approximately 230 residues (Ser617-Arg844) and the resolved portion folds into a complex topology (Figure 3A-G and Figure 6B). A hinge module is formed by proximal (α1, Pro628-Arg640) and distal (α4, Met817-Lys841) α-helical-containing linker elements nucleated around the intracellular S2-S3 loop of VSD3 (Figure 3A, B, D). A lasso-like loop (Pro638-Arg659) emerges from the hinge module to bury multiple hydrophobic side chains across HR-24 and HR-25 on UNC80, followed by a short amphipathic α-helix (α2, Glu660-Arg669) that docks within a conserved surface groove on UNC80 parallel to HR-22 (Figure 3D and F). After disordered extension, the DII-DIII linker emerges at Val711 in the cleft between UNC79 and UNC80, with Arg717 forming the only salt-bridge interaction identified across all three subunits (Figure 3G). Another amphipathic α-helix (α3, Asn718-Ser740) from the DII-DIII linker packs close to the inserted Ub-like domain of UNC80, and then, according to the unresolved structure, the linker connects back to the hinge module at the base of VSD3 (Figure 3A and Figure 3B). Although several linker manipulations did not completely abolish NALCN channelosome function, targeted disruption of the hinge module (ΔAsn829-Asp834) or deletion of the membrane-proximal amphipathic α2-helix excision activity (Figures ​(Figures3I,3J,12C,12D)) identified essential structural components of this critical NALCN-UNC80 interface.

[0127] Example 9. Calmodulin binds to the carboxy-terminal domain of NALCN Our studies unexpectedly identified the C-terminal lobe of CaM bound to a degenerate IQ-like motif (Ile1572-Arg1573) located on the last resolved α-helix of NALCN (Leu1550-Arg1598) immediately below the EF-hand-like domain (Ser1463-Gln1549) in the CTD (Figure 3H). Although less defined in the cryo-EM maps relative to adjacent regions (Figure 6C-D), the C-terminal lobe of CaM is wedged relative to the Ub-like domain of UC80 and HR-20, and CaM binding appears to restrict local contacts between NALCN and UNC80 (Figure 3H). The N-terminal lobe and intralobe linker of CaM are directly adjacent to the EF-hand-like domain of the CTD (Figure 3H). We found that targeted mutations or deletions within the CTD region differentially altered NALCN channelosome currents measured in Xenopus oocytes but did not abolish activity (Figures 3I-J, 12A-G, and 14A-D). In contrast to the NALCN DI-DII and DII-DIII linkers, neither the CTD nor CaM appear to be strictly essential for NALCN channelosome function, although our results indicate that they contribute to the current phenotype.

[0128] Example 10. NALCN channelosomes have an inherently low open probability Apparent constitutive activity of the NALCN channelosome ( Fig. 1A ). 10 Nevertheless, the pore structure is clearly in a nonconducting state, since the S6 gate is narrow and hydrophobically sealed (Figure 4A-B). Indeed, the S6 helix overlaps with a root-mean-square deviation of less than 0.5 Å for the unliganded NALCN-FAM155A subcomplex, revealing a nearly identical closed pore structure (Figure 4B). 24~26 Although intuitively unexpected, these structural observations may reflect the underlying physiology of the NALCN channelosome.

[0129] Cell-attached recordings of NALCN channelosomes transfected into HEK293 cells using slow voltage ramps revealed single-channel conductances of 27.2 + / - 1.1 pS (mean ± sec) and reversal potentials of 5.7 + / - 1.1 mV (mean ± sem) (data not shown). Estimates of single-channel open probability (Po) showed a uniformly low Po (0.04 ± 0.01) across multiple voltages, where a day-by-day plot analysis revealed that the single-trial average Po discretely switched between epochs of no activity and epochs with a Po of approximately 0.1 (data not shown). Thus, the probability of NALCN channelosome opening is low even during periods of high activity, consistent with the observed nonconductive pore structure (Figure 4A).

[0130] We performed single-channel channelosome recordings from patients with the NALCNY578S mutation and found no change in single conductance, but prolonged channel opening and increased Po (data not shown). Daily plot analysis showed that the Y578SNALCN channelosome also switched between inactive and highly active epochs, with increased Po during activity sweeps compared to wild-type NALCN (data not shown).

[0131] Example 11. Linker modifications differentially affect NALCN channelosome gating Careful evaluation of 3D classifications from our NALCN channelosome samples did not identify any reconstructions with an open S6 gate. Instead, two distinct classes emerged that revealed slight lateral rigid-body rotations (~0.5°) of the intracellular UNC79-UNC80 assembly relative to the NALCN pore-forming subunit, with nearly unchanged DII-DIII linker anchor points. This observation leads us to speculate that motion of the UNC79-UNC80 subcomplex relative to NALCN-FAM155A may affect channel gating via physical linkage to the pore.

[0132] After establishing that the degraded DI-DII linker hairpin loop structure (X349-Ala363) is necessary for NALCN channelosome activity, we attempted to modify its binding properties compared to DI-S6 or DIIVSD2. Insertion of a simple 12-residue sequence (SEQ ID NO: 87) before or after the hairpin structure (i.e., [Gly-Gly-Gly-Ser]3) had no effect on the current phenotype of the NALCN channelosome in Xenopus oocytes (Figure 4C and Figure 5E-J). Substitution of the six residues between DI-S6 and the hairpin with a polyproline sequence (P340-P345) had little effect, whereas deletion of these same residues (ΔX340-X345) led to a marked increase in macroscopic current (Figure 4D-E and Figure 13A-B).

[0133] Since the DII-DIII linker is required for NALCN channelosome activity, we also attempted to modify its binding to DII-S6 and DIIIVSD3, respectively. Insertion of all tested [Gly-Gly-Gly-Ser]3 sequences (SEQ ID NO:87) abolished NALCN channelosome activity (Figures 4D-E and 13C-D). Substitution of the six residues between DII-S6 and the hinge module with a polyproline sequence (P621-P626) also abolished the current, whereas deletion of these six residues (ΔX621-X626) significantly increased the current (Figures 4D-E and 13C-D). Taken together, our results indicate that the DII-DIII linker has a prominent role in relaying the putative UNC79-UNC80 movement to NALCNS6 gating, and therefore we expressed isolated DII-DIII linker constructs in trans (Figure 4F-G). A dominant negative effect on channelosome activity was observed in these competition experiments, establishing the DII-DIII linker-UNC80 interface as a critical nexus for NALCN channelosome assembly, gating and regulation (Figure 4F-G).

[0134] Consideration The NALCN-FAM155A-UNC79-UNC80-CaM channelosome structure revealed unexpected features with intriguing physiological implications. UNC79 and UNC80 have emerged as highly specialized auxiliary subunits, and the unique geometry of the UNC79-UNC80 subcomplex likely highlights their early recruitment to the NALCN complex during evolution, rationalizing previous failures to recognize their structural organization as HEAT-repeat proteins. The large interaction surface observed between UNC79 and UNC80 in the subcomplex also confirms previous associations and explains why destabilization or truncation of either subunit can result in NALCN channelosome mislocalization, dysfunction or disease (Figures 2A-H, 2Q-T, and 11A-O). These insights provide a structural basis for understanding why UNC80 disease mutations phenocopy NALCN-based channel pathology. However, it remains unclear whether UNC79 and UNC80 arose by gene duplication, although the incorporation of a Ub-like fold into UNC80 would suggest that this was probably an early event. Furthermore, the unique structure of the UNC79-UNC80 subcomplex raises the speculation that it may provide a scaffolding function for other proteins reported to affect NALCN activity, such as Src kinases or G proteins, or may play an as yet undescribed cellular role.

[0135] CaM has not previously been implicated in the assembly or function of the NALCN channelosome. In our cryo-EM samples, the C-terminal lobe of CaM binds to an IQ-like motif and mediates the assembly or function of the NALCN channelosome. V and Ca VCaM binds to the conserved interface between UNC80 and the NALCN pore-forming subunits, but deletion of the IQ-like motif or C-terminal region of NALCN still allows robust channel activity (Fig. 3I and Fig. S14A-B). In contrast to previous reports, deletion of the NALCN C-terminus prevented the release of extracellular Ca in our experiments. 2+ Regulation by CaM was not affected (Figures S13E-S13F). Our findings do not exclude an important role for the NALCN C-terminus or CaM in regulating gating, but rather point to the possibility of cell-type-specific regulation of the NALCN channelosome.

[0136] Although the NALCN channelosome is constitutively active, the non-conductive pore structure clearly establishes that the hydrophobic S6 region forms the channel gate, even in the presence of FAM155A, UNC79, UNC80 and CaM (Figure 4A-B). Single channel analysis shows a low Po of approximately 0.4, indicating that the channelosomes stay for long periods with no measurable activity, possibly indicating a presumed prolonged closed or inactivated state. These features indicate that the major Na channelosome should have a low basal activity, with the possibility of tightly regulated regulation from external or cellular inputs. + These findings suggest that gain-of-function mutations such as Y578S destabilize the closed S6 gate, increasing Po and increasing the harmful Na + This rationalizes the effect of disease-causing missense mutations clustering around the NALCN pore module, which can cause influx.

[0137] Our study provides insights into the NALCN channelosome assembly and a tentative framework for the pivot beam gating model. FAM155A likely locks onto NALCN from outside the cell and promotes stabilization of the pore-forming subunits. 4、25From the inside of the cell, the UNC79-UNC80 subcomplex docks to the essential DI-DII and DII-DIII linkers of NALCN (Figure 3A-G). The DII-DIII linker forms a relatively rigid contact with UNC80, while the DI-DII linker-UNC79 contact allows a larger range of motion (Figure 4C), and both contacts serve to orient the beam-like structure of UNC79-UNC80 beneath NALCN (Figure 3A-J). For gating, the DII-DIII linker hinge module may function as a pivot point for the rigid-body-like movement of the UNC79-UNC80 assembly relative to the NALCN subunit (Figure 4C and H). In the non-conducting state, since the S6-proximal linker region is disordered in the cryo-EM map (Figure 5F), it lacks sufficient tension on the DI-S6 and DII-S6 helices (Figure 4H), and the S6-gate likely remains closed. The critical role of these linker regions is indicated by their targeted deletion, which results in a gain of function, presumably by increasing the strain of the connection between the S6 gate and the UNC79-UNC80 subcomplex (Figure 4D-E). Thus, we propose a pivot beam model in which the movement of the UNC79-UNC80 subcomplex eventually generates enough tension along the intracellular linker, unbuckling the DI-S6 and DII-S6 helices and allowing the S6 gate to expand for ion conduction (Figure 4H). The collapse of the hydrophobic pore then presumably promotes the re-establishment of the non-conducting state until the movement of the UNC79-UNC80 assembly can again achieve sufficient tension on the S6 gate (Figure 4H). Although speculative, this gating model may also begin to explain previous observations that VSD1 and VSD2 can modify NALCN activity. 10,25 , and suggest how CTD or CaM binding may affect the movement of the UNC79-UNC80 beam-like structure relative to the NALCN-FAM155A pore-forming subcomplex to regulate its activity.

[0138] In summary, the unprecedented structure of the NALCN channelosome highlights its features as an orphan channel in humans and its early evolutionary history within the four-domain ion channel superfamily. Our investigations point to a unique gating mechanism, rationalize the effects of disease-causing mutations in UNC80 and NALCN, and identify maturation sites for selective inhibitor discovery to potentially treat NALCN channelopathy and other electrical disorders. References 1 Lear, BC et al. The ion channel narrow abdomen is critical for neural output of the Drosophila circadian pacemaker.Neuron48,965-976,doi:10.1016 / j.neuron.2005.10.030(2005). 2 Lu, B. et al. The neuronal channel NALCN contributes resting sodium permeability and is required for normal respiratory rhythm.Cell129,371-383,doi:10.1016 / j.cell.2007.02.041(2007). 3 Jospin, M. et al. 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[0139] Table 4 below provides a list of specific sequences referenced herein. Note that for certain peptide segments, such as the NALCNDI-DII and NALCNDII-DIII linker segments (e.g., SEQ ID NOs: 18-30), an N-terminal methionine may be added to the sequences in the table below when constructing an open reading frame for in vivo expression in a host cell. In other instances where peptides may be synthesized in vitro, an N-terminal methionine may not be necessary. For this reason, it is not included in the NALCN linker peptide sequences in the table below. The sequences of certain protein fragments listed in the table below are listed, including an N-terminal methionine before the native portion of the sequence (e.g., SEQ ID NOs: 56 and 58). If a methionine is not necessary to create the fragment, it may be omitted. [Table 4] TIFF2024526284000005.tif255170TIFF2024526284000006.tif255170TIFF2024526284000007.tif254170TIFF2024526284000008.tif255170TIFF2024526284000009.tif253170TIFF2024526284000010.tif253170TIFF2024526284000011.tif253170TIFF2024526284000012.tif253170TIFF2024526284000013.tif253170TIFF2024526284000014.tif253170TIFF2024526284000015.tif253170TIFF2024526284000016.tif253170TIFF2024526284000017.tif253170TIFF2024526284000018.tif253170TIFF2024526284000019.tif255170TIFF2024526284000020.tif253170TIFF2024526284000021.tif255170TIFF2024526284000022.tif255170TIFF2024526284000023.tif255170TIFF2024526284000024.tif255170TIFF2024526284000025.tif255170TIFF2024526284000026.tif255170TIFF2024526284000027.tif255170TIFF2024526284000028.tif255170TIFF2024526284000029.tif255170TIFF2024526284000030.tif255170TIFF2024526284000031.tif255170TIFF2024526284000032.tif255170TIFF2024526284000033.tif255170TIFF2024526284000034.tif255170TIFF2024526284000035.tif255170TIFF2024526284000036.tif255170TIFF2024526284000037.tif255170TIFF2024526284000038.tif255170TIFF2024526284000039.tif255170TIFF2024526284 000040.tif255170TIFF2024526284000041.tif255170TIFF2024526284000042.tif255170TIFF2024 526284000043.tif255170TIFF2024526284000044.tif255170TIFF2024526284000045.tif254170T IFF2024526284000046.tif255170TIFF2024526284000047.tif255170TIFF2024526284000048.tif2 55170TIFF2024526284000049.tif255170TIFF2024526284000050.tif255170TIFF20245262840000 51.tif255170TIFF2024526284000052.tif255170TIFF2024526284000053.tif255170TIFF20245262 84000054.tif255170TIFF2024526284000055.tif255170TIFF2024526284000056.tif255170TIFF20 24526284000057.tif255170TIFF2024526284000058.tif255170TIFF2024526284000059.tif62170.

[0140] The foregoing has been described in some detail by way of detailed description and examples for clarity of understanding, but the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

Claim 1 A method for identifying a modulator of a complex of human NALCN (Na+ leak channel non-selective), FAM155 (family member A of family 155 with sequence similarity), UNC79 (uncoupling 79) and UNC80 (uncoupling 80) (human NALCN-FAM155-UNC79-UNC80 complex), comprising: a) providing the human NALCN-FAM155-UNC79-UNC80 complex in vitro; b) contacting the complex with a potential modulator of the complex; c) performing an ion channel assay on the complex in the presence of the potential modulator; and d) identifying the potential modulator as a modulator of the complex if the activity of the complex in the assay in the presence of the potential modulator is higher or lower than the activity of the complex in the assay in the absence of the potential modulator A method comprising the above steps. Claim 2 The method according to claim 1, wherein the ion channel assay is a patch clamp or automated patch clamp assay, an ion flux assay, or an ion or voltage sensitive dye assay. Claim 3 The method according to claim 1, wherein the activity of the complex in the assay in the presence of the potential modulator is lower than the activity of the complex in the assay in the absence of the potential modulator (i.e., the potential modulator reduces the activity of the complex in the assay). Claim 4 The method according to claim 3, wherein the potential modulator identified in part (c) reduces the activity of the complex in the assay by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Claim 5 The method according to claim 1, wherein the activity of the complex in the assay in the presence of the potential modulator is higher than the activity of the complex in the assay in the absence of the potential modulator (i.e., the potential modulator increases the activity of the complex in the assay). Claim 6 The method according to claim 1, further comprising determining the binding affinity of the potential modulator identified in part (d) for the human NALCN-FAM155-UNC79-UNC80 complex, the human NALCN-FAM155 complex, the UNC79-UNC80 complex, or one or more of human NALCN, FAM155, UNC79, or UNC80. **Claim 7** The method according to claim 6, wherein the potential modulator binds to the human NALCN-FAM155-UNC79-UNC80 complex with an EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less. **Claim 8** The method according to claim 1, wherein the method is performed in the presence of an NALCN-DII-DIII linker peptide or an NALCN-DI-DII linker peptide, or an identified modulator of the human NALCN-FAM155-UNC79-UNC80 complex. **Claim 9** The method according to claim 8, wherein the linker peptide comprises an amino acid sequence of any one of SEQ ID NOs: 16 to 24, or comprises an amino acid sequence of any one of SEQ ID NOs: 16 to 18 or 23 to 24. **Claim 10** The method according to any one of claims 1 to 9, further comprising determining whether the potential modulator modulates the activity of the human NALCN-FAM155-UNC79-UNC80 complex, wherein the complex comprises a mutant human NALCN or a mutant human UNC79 or a mutant human UNC80. **Claim 11** The method according to claim 10, wherein the mutant human NALCN comprises a substitution, insertion, or deletion in one or both of the DI-DII linker or the DII-DIII linker. **Claim 12** a) the DI-DII linker or the DII-DIII linker is replaced by the corresponding region from a human or mammalian Na Ｖ or Ca Ｖ protein; or b) whether the DI-DII linker or the DII-DIII linker contains a deletion; or c) the DI-DII linker or the DII-DIII linker contains an insertion of a GGGGS element, The method according to claim 11. **Claim 13** The method according to claim 10, wherein the mutant human NALCN comprises an amino acid sequence of any one of SEQ ID NOs: 7, 8, 12 to 15, 32 to 52, or 59 to 63. **Claim 14** The method further comprises determining whether the potential modulator modulates the activity of the human NALCN-FAM155-UNC79-UNC80 complex, wherein the complex comprises a mutant human UNC79 comprising any one amino acid sequence of, for example, SEQ ID NO: 53, 54, 55, 58 or 64-73, the method according to any one of claims 1-9.

15. The method further comprises determining whether the potential modulator modulates the activity of the human NALCN-FAM155-UNC79-UNC80 complex, wherein the complex comprises a mutant human UNC80 comprising any one amino acid sequence of, for example, SEQ ID NO: 74-85, the method according to any one of claims 1-9.

16. A method for identifying a molecule that binds to a complex of human NALCN (Na+ leak channel non-selective), human FAM155 (family with sequence similarity 155 member A), human UNC79 (uncoupled 79), and human UNC80 (uncoupled 80): (human NALCN-FAM155-UNC79-UNC80 complex), comprising: a) providing a human NALCN-FAM155-UNC79-UNC80 complex in vitro; b) contacting the complex with one or more test molecules and separating the bound test molecules from the unbound test molecules; and c) identifying the test molecule as a molecule that binds to the human NALCN-FAM155-UNC79-UNC80 complex if the test molecule remains bound to the complex after separation from the unbound test molecules. A method comprising.

17. A method for identifying a molecule that binds to a complex of human NALCN (Na+ leak channel non-selective), human FAM155 (family with sequence similarity 155 member A), human UNC79 (uncoupled 79), and human UNC80 (uncoupled 80): (human NALCN-FAM155-UNC79-UNC80 complex), comprising: a) providing a human NALCN-FAM155-UNC79-UNC80 complex in vitro; b) contacting the complex with a NALCN-DII-DIII linker peptide or a NALCN-DI-DII linker peptide, and further contacting with a test molecule; and c) Identifying the test molecule as a molecule that binds to the human NALCN-FAM155-UNC79-UNC80 complex when the test molecule competes with the NALCN-DII-DIII linker peptide and / or the NALCN-DI-DII linker peptide for binding to the complex A method comprising the above. **Claim 18** A method for identifying a molecule that regulates the in vitro formation or stability of a complex of human NALCN (Na+ leak channel non-selective), human FAM155 (family member A of family 155 with sequence similarity), human UNC79 (uncoupled 79), and human UNC80 (uncoupled 80): (human NALCN-FAM155-UNC79-UNC80 complex), comprising: a) Providing human NALCN, human FAM155, human UNC79, and human UNC80 under conditions where the formation of the human NALCN-FAM155-UNC79-UNC80 complex is promoted in vitro; b) Contacting the complex with a test molecule; and c) Identifying the test molecule as a molecule that regulates the formation or stability of the human NALCN-FAM155-UNC79-UNC80 complex when the degree of complex formation in the presence of the test molecule is increased or decreased compared to in the absence of the test molecule A method comprising the above. **Claim 19** The method according to any one of claims 16 to 18, wherein the potential modulator binds to the human NALCN-FAM155-UNC79-UNC80 complex with an EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less. **Claim 20** The method according to any one of claims 16 to 18, further comprising determining the binding affinity of the test molecule for the complex by an ELISA, AlphaLISA, or FRET assay. **Claim 21** The method according to any one of claims 1 to 9, wherein at least one of the human NALCN, human FAM155, human UNC79, or human UNC80 of the human NALCN-FAM155-UNC79-UNC80 complex is labeled. **Claim 22** The method according to any one of claims 1 to 9, wherein at least one of the human NALCN, human FAM155, human UNC79, or human UNC80 of the human NALCN-FAM155-UNC79-UNC80 complex is labeled and / or bound to a matrix.

23. The method according to any one of claims 1 to 9, wherein the human NALCN-FAM155-UNC79-UNC80 complex is solubilized in a lipid bilayer, a surfactant, or a lipid nanodisc.

24. The method according to any one of claims 16 to 18, further comprising determining whether the molecule binds to a human NALCN-FAM155 complex, a human UNC79-UNC80 complex, or one or more of human NALCN, FAM155, UNC79, or UNC80.

25. The method according to any one of claims 16 to 18, further comprising determining whether the test molecule binds to a human NALCN-FAM155-UNC79-UNC80 complex, wherein the complex comprises a mutant human NALCN.

26. The method according to claim 25, wherein the mutant human NALCN comprises a substitution, insertion, or deletion in one or both of the DI-DII linker or the DII-DIII linker. **Claim 27** a) whether the DI-DII linker or the DII-DIII linker is replaced by the corresponding loop from a human or mammalian Na Ｖ or Ca Ｖ protein; b) whether the DI-DII linker or the DII-DIII linker comprises a deletion; or c) the DI-DII linker or the DII-DIII linker comprises an insertion of a GGGGS element, The method according to claim 26.

28. The method according to claim 26, wherein the mutant human NALCN comprises an amino acid sequence of any one of SEQ ID NOs: 7, 8, 12-15, 32-52, or 59-63.

29. The method according to any one of claims 1 to 9, wherein the human NALCN-FAM155-UNC79-UNC80 complex further comprises human calmodulin.

30. The method according to any one of claims 1 to 9, wherein the potential modulator or test molecule is a peptide, a macrocyclic polymer, or an antibody.

31. The method according to any one of claims 1 to 9, wherein the potential modulator or test molecule is a small molecule.

32. A molecule identified by the method according to claim 1.

33. The molecule according to claim 32, which is a peptide, a macrocyclic polymer, or an antibody.

34. The molecule according to claim 32, which is a small molecule.

35. An identified peptide modulator of human NALCN, comprising any one of the amino acid sequences of SEQ ID NOs: 16 to 18 or 23 to 24.

36. The molecule or the identified peptide modulator according to any one of claims 33 to 35, for use in treating channelopathies, neurodevelopmental disorders, circadian rhythm disorders, or pain in a subject.

37. A method of treating a subject having a channelopathy, a neurodevelopmental disorder, a circadian rhythm disorder, or pain, the method comprising administering to the subject an effective amount of the molecule or the identified peptide modulator according to any one of claims 33 to 35.

38. An identified modulator of the human NALCN-FAM155-UNC79-UNC80 complex, one or more reagents for performing an ion channel assay, a labeled NALCN, UNC79, UNC80, or FAM155, and instructions for use, comprising at least one of A kit for performing the method according to any one of claims 1 to 9.

39. The kit according to claim 38, wherein the labeled NALCN, UNC79, UNC80, or FAM155 is bound to a matrix selected from beads, chips, and plates.

40. An isolated complex of human NALCN, UNC79, UNC80, and FAM155.

41. The isolated complex according to claim 40, wherein the complex is contained within a lipid bilayer or a lipid nanodisc.

42. a) at least one of the human NALCN, UNC79, UNC80, and FAM155 contains a label; or b) at least one of the human NALCN, UNC79, UNC80, and FAM155 is bound to a matrix. The isolated complex according to claim 40 or 41.

43. The isolated complex according to claim 40, further comprising a modulator of human NALCN or a molecule identified by the method according to any one of claims 1 to 9.

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