Methods and compositions for measuring membrane channel activity

Abstract

Methods and compositions for embedding structures which are channels, hemichannels, pores, or have other structurally defined openings inside of a synthetic cell are discussed. These synthetic cells can be a vesicle. These synthetic cells with embedded structures can be used for a variety of detection and screening methods.

Description

METHODS AND COMPOSITIONS FOR MEASURING MEMBRANE CHANNEL ACTIVITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U. S. Provisional Application No. 63 / 712,786, filed October 28, 2024, incorporated herein by reference in its entirety.BACKGROUND

[0002] In mammalian cells, a class of membrane proteins called connexins self-associate to form hexameric nanopores called hemichannels. These hemi channels have the unique ability to pair with other such hemichannels (HC) on neighboring cells to form tightly sealed, doublemembrane intercellular channels. These channels, called connexin channels, fill the gap between adjacent cells and facilitate the direct transfer of molecules between neighboring cytoplasm. Under normal physiological conditions, unpaired HCs remain primarily in a closed state, thereby ensuring that intercellular communication proceeds through only connexin channels. However, pathological conditions result in active HCs, which in turn exacerbates many disorders. HC activation has been implicated in several neurodegenerative conditions, such as Alzheimer’s disease, cerebral ischemia, amyotrophic lateral sclerosis, vasculopathies and cancer. This makes active connexin HCs an attractive therapeutic target.

[0003] What is needed in the art are methods and assays for identifying direct HC inhibitors, which leads to improved treatment options. Such efforts have been hampered by a lack of effective screening platforms that can assay HC function in a high-throughput fashion. These, and other important needs, are satisfied by the present disclosure.SUMMARY

[0004] Disclosed herein is a composition comprising a synthetic cell system, wherein said system comprises: a) at least one vesicle mimicking a cell membrane; b) at least one structure embedded within the vesicle, wherein the structure comprises an opening or channel, wherein the opening or channel can allow material to pass from one side of the membrane to another; and c) at least one detectable probe.

[0005] Also disclosed is a method of determining that an analyte affects transport of a molecule across a pore / channel, the method comprising: a) providing a synthetic cell system comprising: i) at least one vesicle mimicking a cell membrane, ii) at least one structureembedded within the vesicle, wherein the structure comprises an opening or channel, wherein the opening or channel can allow material to pass from one side of the membrane to another, iii) at least one detectable probe, and a substance which can react with the probe on an opposite side of the vesicle than the detectable probe; b) exposing the analyte to the synthetic cell system; and c) measuring a change in signal from the detectable probe, wherein a difference in signal indicates that the analyte affects transport of the molecule.

[0006] Further disclosed is a method of making a synthetic cell system with a pore or channel therein, the method comprising providing a plasmid which encodes for a modified channel, hemi-channel, or pore-forming protein in presence of at least one vesicle mimicking a cell membrane, wherein said modified channel, hemi-channel, or pore-forming protein is capable of insertion into the vesicle once it is expressed.

[0007] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0009] Figure 1 shows cell communication across gap junctions.

[0010] Figure 2 shows a schematic of connexin hemichannels as therapeutic targets, and how normal tissue functions.

[0011] Figure 3 shows connexin hemi channels as therapeutic targets, and how diseased tissue functions, where the hemichannels are overactivated, and a connexin channel is not functional.

[0012] Figure 4 shows a schematic of a synthetic cell-based assay.

[0013] Figure 5 shows a bar chart which shows fluorescence readings for different experimental conditions were recorded using a plate reader. The first condition (-pCx43) performed using synthetic cells lacking connexin-43 (Cx43) hemichannels shows minimal fluorescence turn on of the probe. However, a significant increase in fluorescence is observed in synthetic cells that contain hemichannels (+pCx43). Addition of different Cx 43 hemichannel inhibitors, which are- Gap26 (peptide), CBX (carbenoxolone, small molecule), Ca2+(ion),9renders the hemichannel inactive and as a consequence, we see a marked decrease in fluorescence.

[0014] Figure 6 shows a bar chart which shows fluorescence readings for different experimental conditions that were recorded using a plate reader. The first condition (-pCx43) performed using synthetic cells lacking connexin-43 (Cx43) hemichannels shows minimal fluorescence turn on of the probe (CalFluor 488). However, a significant increase in fluorescence is observed in synthetic cells that contain hemichannels (+pCx43).DETAILED DESCRIPTION

[0015] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0016] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0017] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0018] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a disease”, includes, but is not limited to, two or more such compounds, compositions, or diseases, and the like.

[0019] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0020] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0021] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1%’ to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0022] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are notand need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0023] Amino acids used in compounds provided herein (e.g. peptides and proteins) can be genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids. Both L- and D-enantiomers of any of the above can be utilized in the compounds. The following abbreviations may be used herein for the following genetically encoded amino acids (and residues thereof): alanine (Ala, A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine (Cys, C); glycine (Gly, G); glutamic acid (Glu, E); glutamine (Gin, Q); histidine (His, H); isoleucine (He, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Vai, V).

[0024] Certain commonly encountered amino acids that are not genetically encoded and that can be present in the compounds of the invention include, but are not limited to, P-alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr, Z), 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib); s-aminohexanoic acid (Aha); 6-amino valeric acid (Ava); methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle, J); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3 -fluorophenyl alanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal); homocysteine (hCys); 3-benzothiazol-2-yl-alanine (BztAla, B); and homoserine (hSer). Additional amino acid analogs contemplated includephosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, a-methyl-alanine, para-benzoyl-phenylalanine, propargylglycine, and sarcosine. Peptides that are encompassed within the scope of the invention can have any of the foregoing amino acids in the L- or D-configuration, or any other amino acid described herein or known in the art, whether currently or in the future.

[0025] Amino acids that are substitutable for each other generally reside within similar classes or subclasses. As known to one of skill in the art, amino acids can be placed into different classes depending primarily upon the chemical and physical properties of the amino acid side chain. For example, some amino acids are generally considered to be hydrophilic or polar amino acids and others are considered to be hydrophobic or nonpolar amino acids. Polar amino acids include amino acids having acidic, basic or hydrophilic side chains and nonpolar amino acids include amino acids having aromatic or hydrophobic side chains. Nonpolar amino acids may be further subdivided to include, among others, aliphatic amino acids.

[0026] “Anti-connexin compounds” include those compounds that affect or modulate the activity, expression or formation of a connexin, a connexin hemichannel (connexon), or a gap junction. Anti-connexin compounds include without limitation antisense compounds (e.g. antisense polynucleotides), antibodies and binding fragments thereof, and peptides and polypeptides which include “peptidoniimetic” and “mimetic” peptides. Any of the embedded structures described herein can have a compound which specifically targets it, and anti-connexins are provided by way of example.

[0027] “Antisense compounds” include different types of molecules that act to inhibit gene expression, translation, or function, including those that act by sequence- specific targeting of mRNAs for therapeutic applications. Antisense compounds include antisense DNA compounds and antisense RNA compounds. While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e., from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. They also include phosphorothioate oligodeoxynucleotides (S-ODNs).

[0028] Antisense compounds thus include, for example, the major nucleic-acid based genesilencing molecules such as, for example, chemically modified antisense oligodeoxyribonucleic acids (ODNs), ribozymes and siRNAs (Scherer, L. J. and Rossi, J. J. Nature Biotechnol. 21: 1457-1465 (2003). Antisense compounds may also include antisense molecules such as, for example, peptide nucleic acids (PNAs) (Braasch, D. A. and Corey, D. R., Biochemistry 41, 4503-4510 (2002)), morpholino phosphorodiamidates (Heasman, J., Dev. Biol., 243, 209-214 (2002), DNAzymes (Schubert, S. et al., Nucleic Acids Res. 31, 5982-5992 (2003). Chakraborti, S. and Banerjea, A. C., Mol. Ther. 7, 817-826 (2003), Santoro, S. W. and Joyce, G. F. Proc. Natl Acad. Sci. USA 94, 4262-4266 (1997), and the recently developed 5 ’ -end-mutated U1 small nuclear RNAs (Fortes, P. et al., Proc. Natl. Acad. Sci. USA 100, 8264-8269 (2003).

[0029] In general, nucleic acids (including oligonucleotides, discussed below) may be described as “DNA-like” (i.e., having 2’ -deoxy sugars and, generally, T rather than U bases) or “RNA-like” (i.e., having 2’-hydroxyl or 2’-modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are “DNA-like” and those which have A-form-like structure are “RNA-like.”

[0030] The term “complementary” generally refers to the natural binding of polynucleotides by base pairing, for example under permissive salt and temperature conditions. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A”. Complementarity between two single-stranded molecules may be “partial”, such that only some of the nucleic acids bind, or it may be “complete”, such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid molecules has significant effects on the efficiency and strength of the hybridization between them. “Hybridizable” and “complementary” are terms that are used to indicate a sufficient degree of complementarity such that binding, preferably stable binding sufficient to carry out an intended action, for example, occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be hybridizable, and it is also understood that the binding may be target-specific, or may bind to other non-target molecules so long as the non-specific binding does not significantly or undesirably thwart the therapeutic or other objective. An oligonucleotide is used to interfere with the normal function of the target molecule to cause a loss or diminution of activity, and it is preferred that there is a sufficient degree ofcomplementarity to avoid non-specific or unwanted binding of the oligonucleotide to nontarget sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. Absolute complementarity is not required. Polynucleotides that have sufficient complementarity to form a duplex having a melting temperature of greater than 20° C, 30° C, or 40° C under physiological conditions, are generally preferred.

[0031] A “disorder” is any condition that would benefit from treatment with a molecule or composition of the invention, including those described or claimed herein. This includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question.

[0032] As used herein, “subject” refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc. The preferred subject is a human.

[0033] The term “oligonucleotide” includes an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term “oligonucleotide” also includes oligomers or polymers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, increased stability in the presence of nucleases, or enhanced target affinity. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide (ODN). Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides, which have been modified to enhance their nuclease resistance, can survive intact for a longer time than unmodified oligonucleotides. A number of modifications have also been shown to increase binding (affinity) of the oligonucleotide to its target. Affinity of an oligonucleotide for its target is routinely determined, for example, by measuring the Tm (melting temperature) of an oligonucleotide / target pair, which is the temperature at which the oligonucleotide and target dissociate. Dissociation may be detected spectrophotometrically. The greater the I'm, the greater the affinity of the oligonucleotide has for the target. In some cases, oligonucleotide modifications which enhance target-binding affinity are also able to enhance nuclease resistance.

[0034] A “polynucleotide” means a plurality of nucleotides. Thus, the terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” or “oligodeoxynucleotide” all refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.

[0035] A polynucleotide that encodes a structure to be embedded, such as a connexin, a connexin fragment, or a connexin variant includes a polynucleotide encoding: the mature form of the connexin found in nature (and naturally occurring and species variants thereof); the mature form of the connexin found in nature and additional coding sequence, for example, a leader or signal sequence or a proprotein sequence (and naturally occurring and species variants thereof); either of the foregoing and non-coding sequences (for example, introns or non-coding sequence 5’ and / or 3’ of the coding sequence for the mature form(s) of the polypeptide found in nature); fragments of the mature form(s) of the connexin found in nature; and, as noted, variants of the mature form(s) of the connexin found in nature. Thus, “connexin-encoding polynucleotide” and the like encompass polynucleotides that have only a coding sequence for a desired connexin, fragment, or variant, as well as polynucleotides that includes other nucleotides such as additional coding and / or non-coding sequences. This applies to not only connexins, but all embedded structures.

[0036] The terms “peptidomimetic” and “mimetic” include naturally occurring and synthetic chemical compounds that may have substantially the same structural and functional characteristics of protein regions which they mimic. For connexins these may mimic, for example, the extracellular loops of opposing connexins involved in connexon-connexon docking and cell-cell channel formation.

[0037] Peptide analogs with properties analogous to those of the template peptide may be non-peptide drugs. “Peptide mimetics” or “peptidomimetics,” which include peptide -based compounds, also include such non-peptide based compounds (Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger; TINS; 392 (1985); and Evans et al., J. Med. Chem. 30: 1229 (1987); Beeley N., Trends Biotechnol. June; 12(6): 213-6 (1994); Kieber-Emmons T, et al.; Corr Opin Biotechnol. August; 8(4): 435-41 (1997). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally identical or similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological function or activity), but can also have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of, for example, — CH2NH —, — CH2. S —, — CH2-CH2-, — CH— CH— (cis and trans), — COCH2-, — CH(OH)CH2-, and — CH2SO—. The mimetic can be either entirely composed of natural amino acids, or non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly nonnatural analogs of amino acids. The mimetic can also comprise any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter mimetic activity. For example, a mimetic composition is within the scope of the invention if it is capable of down-regulating biological actions or activities of connexin proteins or connexons, such as, for example, preventing the docking of connexons to form gap-junction-mediated cell-cell communication, or preventing the opening connexons to expose the cell cytoplasm to the extracellular milieu. Peptidomimetics, mimetic peptides, and connexin modulating peptides encompass those described such peptidomimetics, mimetic peptides, and connexin modulating peptides set forth herein, as well as those as may be known in the art, whether now known or later developed.

[0038] The term “composition” is intended to encompass a product comprising one or more ingredients.

[0039] The terms “modulator” and “modulation” of an embedded structure, such as connexin activity, as used herein in its various forms, is intended to encompass inhibition in whole or in part of the expression or action or activity of a connexin. Such modulators include small molecules antagonists of connexin function or expression, antisense molecules, ribozymes, triplex molecules, and RNAi polynucleotides, gene therapy methods, etc., and others.

[0040] The phrase “percent (%") identity” refers to the percentage of sequence similarity found in a comparison of two or more sequences. Percent identity can be determined electronically using any suitable software, for example. Likewise, “similarity” between two sequences (or one or more portions of either or both of them) is determined by comparing the sequence of one sequence to a second sequence.

[0041] “Pharmaceutically acceptable” compounds and other ingredients of a composition or formulation, for example, a carrier, diluent or excipient, are those that are suitable for administration to a recipient thereof.

[0042] In general, the term “protein” refers to any polymer of two or more individual amino acids (whether or not naturally occurring) linked via peptide bonds, as occur when the carboxyl carbon atom of the carboxylic acid group bonded to the alpha-carbon of one amino acid (oramino acid residue) becomes covalently bound to the amino nitrogen atom of the amino group bonded to the alpha-carbon of an adjacent amino acid. 'These peptide bond linkages, and the atoms comprising them (i.e., alpha-carbon atoms, carboxyl carbon atoms (and their substituent oxygen atoms), and amino nitrogen atoms (and their substituent hydrogen atoms)) form the “polypeptide backbone’’ of the protein. In addition, as used herein, the term “protein” is understood to include the terms “polypeptide” and “peptide” (which, at times, may be used interchangeably herein). Similarly, protein fragments, analogs, derivatives, and variants are may be referred to herein as “proteins,” and shall be deemed to be a “protein” unless otherwise indicated. The term “fragment” of a protein refers to a polypeptide comprising fewer than all of the amino acid residues of the protein. A “domain” of a protein is also a fragment, and comprises the amino acid residues of the protein often required to confer activity or function.

[0043] The term “therapeutically effective amount” means the amount of the subject compound that will elicit a desired response, for example, a biological or medical response of a tissue, system, animal or human that is sought, for example, by a researcher, veterinarian, medical doctor, or other clinician.

[0044] “Treatment” refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0045] The term “vector” refers to a nucleic acid molecule amplification, replication, and / or expression vehicle in the form of a plasmid, phage, viral, or other system (be it naturally occurring or synthetic) for the delivery of nucleic acids to cells where the plasmid, phage, or virus may be functional with bacterial, yeast, invertebrate, and / or mammalian host cells. The vector may remain independent of host cell genomic DNA or may integrate in whole or in part with the genomic DNA. The vector will generally but need not contain all necessary elements so as to be functional in any host cell it is compatible with. An “expression vector” is a vector capable of directing the expression of an exogenous polynucleotide, for example, a polynucleotide encoding a binding domain fusion protein, under appropriate conditions.

[0046] As described herein, the terms “homology and homologues” include polynucleotides that may be a homologue of sequence in connexin polynucleotide (e.g. mRNA). Such polynucleotides typically have at least about 70% homology, preferably at least about 80%, 90%, 95 %, 97% or 99% homology with the relevant sequence, for example over a region of at least about 15, 20, 30, 40, 50, 100 more contiguous nucleotides (of the homologous sequence).

[0047] The term “gene product” refers to an RNA molecule transcribed from a gene, or a polypeptide encoded by the gene or translated from the RNA.

[0048] The term “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (for example, “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. Thus, a “recombinant” polynucleotide is defined either by its method of production or its structure. In reference to its method of production, the process refers to use of recombinant nucleic acid techniques, for example, involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a polynucleotide made by generating a sequence comprising a fusion of two or more fragments that are not naturally contiguous to each other. Thus, for example, products made by transforming cells with any non-naturally occurring vector is encompassed, as are polynucleotides comprising sequence derived using any synthetic oligonucleotide process. Similarly, a “recombinant” polypeptide is one expressed from a recombinant polynucleotide.GENERAL DESCRIPTIONMembrane Transport and Conn exins

[0049] Membrane transport is a fundamental means to control basic cellular processes such as apoptosis, inflammation, and neurodegeneration and is mediated by a number of transporters, pumps, and channels (Syrjanen 2021). Large pore channels exist in various tissues and organs in gap-junctional and non-gap-junctional forms in order to flow various metabolites and ions. They are formed by a number of protein families which are described herein. Connexins, one of these protein families, constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells.

[0050] Gap junctions are specialized intercellular connections that are found between most animal cell -types. They are expressed in virtually all tissues of the body, with the exception of mature skeletal muscle and mobile cell types such as sperm and erythrocytes. Gap junctions directly connect the cytoplasm of two cells, which allows various molecules, ions and electrical impulses to directly pass through a regulated gate between cells.

[0051] One gap junction channel can be composed of two connexons (or hemichannels), which connect across the intercellular space between adjacent cells and allow intracellular molecules to flow between those cells. Each connexon of a gap junction resides in the adjacentcell membrane and is formed by the covalent oligomerization of six individual connexins (“Cx”, or “Cxn”) proteins (Yeager 1998, Dbouk 2009).

[0052] Connexin proteins and their associated gap junction channels come in a range of sizes and configurations that are thought to offer some specificity for the chemical species that can pass through. All connexins share a common structure with four transmembrane domains, two extracellular loops, a cytoplasmic loop, a short cytoplasmic amino terminus and a carboxy terminus that can vary considerably in length (Unger 1999). The connexin proteins are commonly named according to their molecular weights, e.g., Cx26 is the connexin protein of 26 kDa.

[0053] The function and / or dysfunction of gap junctions has been implicated in a number of disorders. For example, connexin30 is mutated in Clouston syndrome (liidrotic ectodermal dysplasia), and mutations in the connexin26 gene are the most common cause of genetic deafness. Mutations in the human connexin32 gene cause X-linked Charcot-Marie-Tooth disease, a hereditary neuropathy, while oculodentodigital dysplasia is generally believed to be caused by a mutation in the gene that codes for connexin43. A list of human connexins and their associated gene and examples of diseases or disorders associated with that connexin are listed below in Table 1.Table 1: Human connexins and clinical significance

[0054] Gap junctions are essential for many physiological processes, such as the coordinated depolarization of cardiac muscle, proper embryonic development, and the conducted response in microvasculature. For this reason, deletion or mutation of the various connexin isoforms produces distinctive phenotypes and pathologies. While mutations in Cx47are associated with Pelizaeus-Merzbacher-like disease and lymphedema, Cx40 mutations are principally linked to atrial fibrillation. Mutations in Cx37 have not yet been described, but polymorphisms in the Cx37 gene have been implicated in the development of arterial disease.

[0055] Pannexins are a family of transmembrane channel glycoproteins that include Panx1, Panx2 and Panx3. Pannexins share similar structural features with connexins, consisting of 4 transmembrane domains, 2 extracellular and 1 intracellular loop, along with intracellular N- and C-terminal tails. Panx1 is expressed in many mammalian tissues, while Panx2 and Panx3 expression is more limited. Panxl has been linked to propagation of calcium waves, regulation of tone, mucociliary lung clearance, and taste -bud function. Panxl is expressed in the brain, bladder, testis, and ovary, whereas Panx2 is expressed primarily in the brain, and Panx3 is expressed in skin, cartilage, heart, kidney and cochlea. Panxl hemichannels have been implicated in ATP release, calcium signaling, keratinocyte and osteoblast differentiation, taste reception, cell death, post-ischemic neurodegeneration, tumor suppression and seizure. Panx2 is involved in differentiation of neurons while Panx3 plays a role in the differentiation of chondrocytes, osteoblasts and the maturation and transport of sperm. Panxl is localized to the plasma membrane whereas Panx2 is intracellularly located. One major difference between connexin and pannexin channels is that pannexin channels do not form cell-to-cell channels and it has been suggested that the highly glycosylated extracellular loops of pannexin proteins interferes with the docking process. As with connexin hemichannels, pannexin channels are said to be activated by a number of factors, but also show some differences. Both connexin hemichannels and pannexin channels contribute to glutamate and ATP release, although pannexin channels are insensitive to decreases in calcium ion concentration (US Patent Application No. US20200239889A1).Synthetic Cells (Vesicles) Modified with an Embedded Structure

[0056] As discussed in the background section, a model of a synthetic cell system that comprises openings or channels which can be used as a model is much needed in the art. This model can allow for testing or screening of the interactivity of various compounds and compositions on the channels, openings or pores. It can also be used to study the interaction of channels, openings, or pores with each other and with other channels, openings or pores.

[0057] Disclosed herein is a composition comprising a synthetic cell system, wherein said system comprises: a) at least one vesicle mimicking a cell membrane; b) at least one structure embedded within the vesicle, wherein the embedded structure comprises an opening or channel, wherein the opening or channel can allow material to pass from one side of themembrane to another; and c) at least one detectable probe. This system can be seen in Figure 4, for example.

[0058] Lipid vesicles, or liposomes, which mimic cell membranes in structure and components have been widely used to study the origin of life, artificial cell construction, and drug delivery carriers (Suzuki 2022). The use of lipid vesicles promotes an understanding of essential cellular functions, including membrane protein functions, membrane fusion and fission, complex enzymatic reactions, and protein expression. V esicle-based artificial cells are discussed in Lu et al. (2022), Xu et al. (2016), and Shin et al. (2022), all of which are herein incorporated by reference in its entirety.

[0059] The synthetic cell which can be used with the invention can comprise a vesicle which is composed of lipid material, polymeric material, extracted native cellular material, or a hybrid of any of these. Specifically, the lipid vesicle can comprise a phospholipid bilayer, as naturally found in a cell. The vesicle can further comprise additional components as well, which can be engineered as the synthetic cell is made, or can be added later by exposure of the component to the cell.

[0060] The opening or channel (referred to herein as the “embedded structure”) which is expressed in the synthetic cell can be open, closed, or semi-open. Examples of these states can be seen in Figures 2 and 3. The embedded structure can comprise a protein. This protein can be naturally-occurring, or can be engineered so that it differs from a naturally occurring protein. For example, the engineered protein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more variants compared to the naturally occurring protein. These variants can be additions, substitutions, or deletions of amino acids or to the nucleic acids which encode them.

[0061] The embedded structure (opening or channel) can comprise a hemi -channel or pore-forming protein. Examples of these potentially embedded structures include, but are not limited to, connexin, innexin, pannexin, leucine-rich repeat-containing 8 (LRRC8), calcium homeostasis, modulator (CALHM), porin, voltage-gated ion channels (Na+, Ca2+, K+, CT), ligand-gated ion channels (such as nicotinic acetylcholine receptors (nAChR), y-aminobutyric acid (GABA) receptors, 5-hydroxytryptamine-3 (5HT3) receptors, and glycine receptors), ionotropic glutamate receptors, P2X receptors, mechano-sensitive ion channels, or engineered or non-naturally occurring pore-forming proteins. It is noted that the detectable probe can be chosen based on pore size, such that a specific probe or detection system can be used which allows for the molecule of choice to pass through the embedded structure for detection purposes. For example, for voltage- and ligand-gated ion channels, since the pore size is fairly small, a fluorogenic system can be designed that “fits” within that channel.

[0062] The “embedded structures” can be from a variety of organisms, such as bacteria, fungi, sea anemones, honey bees, etc. Examples include, but are not limited to, hemolysins, cytolysins, aerolysins, D4 domain of the Wza polysaccharide transporter, actinoporins, and melittin. Synthetically designed de novo protein nanopores also fall under the “embedded structures.” In fact, the “embedded structures” also include completely synthetic nanopores, including but not limited to synthetic biomolecule structures and polymeric structures, or hybrids of biomolecule nanopores that are chemically modified with non-natural moieties.(See Margheritis et al. Int J Mol Sci. 2023 Feb 25;24(5):4528 and Ayub et al. Current Opinion in Chemical Biology 34:117 2016, both of which are herein incorporated by reference in their entirety).

[0063] A variety of probes can be used with the present invention. For example, a fluorogenic dye can be used. Examples of fluorogenic dye include, but are not limited to, CalFluors and Heidelberg dyes. In addition, molecules that produce a signal upon binding an “activating” biomolecule (e.g., proteins, DNA, RNA, biosensors, etc.) can also be used as probes. For e.g., modified thiazole orange, malachite green, and 4-hydroxybenzylidene-rhodanine derivatives are all fluorogens that can be compatible with the current invention. Further, the signal produced upon the interaction of the probe and its partner biomolecule, including but not limited to split enzymes, can be read-out as a change in luminescence, absorbance, optical polarization, etc., and is not limited to fluorescence. (See Xu et al. Acta Pharm Sin B. 2018 May;8(3):339-348 and Plamont et al. Proc Natl Acad Sci U S A. 2016 Jan 19; 113(3):497-502; herein incorporated by reference in their entirety).

[0064] In a specific embodiment, the probe can be provided on either side of the synthetic cell’s membrane. If the embedded structure (pore, opening, or channel) is open, the probe can traverse through the opening and can then be detected on the other side (opposite side) of the membrane. For example, the probe can initially be on the outside of the membrane. If the embedded structure is open (traversable), the probe can cross the membrane through the opening and can be detected on the inside (inner portion) of the synthetic cell’s membrane. This can be seen, for example, in Figure 4.

[0065] The probe can have a reactive partner on the other side of the membrane. That way, when the probe crosses the membrane through the embedded structure, it can then react with the partner, which will allow for its detection. This reactive partner can cause either an increase or a decrease in measurable signal from the probe. A variety of probe / reactive partner combinations can be used with the invention. Exposure of the probe to the reactive partner can cause a change in signal of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%-, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times or more compared to a separation of the probe and reactive partner, or compared to a control in which one or the other of the probe or the reactive partner are not present.

[0066] A specific example of a reactive partner includes, but is not limited to, dibenzocyclooctyne (DBCO). For example, the vesicle (synthetic cell) can be loaded with DBCO-modified casein. Another embodiment includes, but is not limited to, DBCO-sulfo-NHS ester. DBCO can be found inside the synthetic cell membrane, for example, while the fluorogenic molecule can be placed outside of the membrane. In one example, the fluorogenic molecule is quenched, or inhibited, until it has crossed the membrane. The reactive partner may be prevented from crossing the membrane by size or other electrophysical or chemical properties which prevent it from traversing through the opening. This way, only the probe can traverse the opening. Once in contact with each other, the probe and the reactive partner can react by click chemistry, thereby producing a detectable signal. It is noted that this is just one example, but there are a variety of ways to produce a detectable signal.

[0067] A variety of means of probe detection, or probe-reactive partner interaction detection, are known to those of skill in the art. Examples include fluorescent microscopy, signal detection through a microplate reader, Fourier transform infrared spectroscopy (FTIR), flow cytometry, and running native sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using a plate reader can be easily done to analyze multiple samples in parallel.

[0068] The synthetic cell / embedded structure system disclosed herein is highly scalable and can be used in a multiplex fashion. For example, more than one vesicle (synthetic cell) can be used in the assay. 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1,000, 10,000, 100,000 or more synthetic cells can be used. The assay can be a high-throughput assay. Each different vesicle can comprise the same embedded structure (channel or opening) or can comprise different structures. One vesicle can comprise more than one structure. These embedded structures can be the same, or can be different than each other. More than one probe can be used simultaneously, and more than one reactive partner can be used simultaneously. The synthetic cells can be on a plate or in any other format where more than one vesicle can be present.Screening Methods and Assays

[0069] Also disclosed herein is a method of determining that an analyte affects transport of a molecule across a pore / channel, the method comprising: a) providing a synthetic cell system comprising: i) at least one vesicle mimicking a cell membrane, ii) at least one structure embedded within the vesicle, wherein the embedded structure comprises an opening or channel, wherein the opening or channel can allow material to pass from one side of the membrane to another, iii) at least one detectable probe, and iv) a substance which can react with the probe on an opposite side of the vesicle than the detectable probe; b) exposing the analyte to the synthetic cell system; and c) measuring a change in signal from the detectable probe, wherein a difference in signal indicates that the analyte affects transport of the molecule.

[0070] In this method, a test analyte (or group of different analytes) is tested to determine if they have an effect on the embedded structure (pore or channel opening). This can be done by comparing the flow rate, or uptake rate or amount of uptake of the analyte compared to a control. If the test analyte causes a change in the amount or rate at which substances can cross the membrane, then it can be determined that the test analyte is capable of modulating the embedded structure, or at least modulating the flow of molecules across the embedded structure. This can be used as a form of drug discovery to determine whether a given analyte can be used to control an opening, channel, or pore, so that it can remain open longer or have a greater width, or allow for the passage of more molecules through it, or allow for the passage of different molecules through it, or allow for a different rate of passage of molecules through it. Conversely, the analyte may have a deleterious effect on the opening, channel or pore, such that it remains open for a shorter duration, or has a narrower width, or allows the passage of less molecules through it, or doesn’t allow for the passage of as large of a variety of molecules through it.

[0071] Also disclosed is a method of making a synthetic cell system with a pore or channel therein, the method comprising providing a plasmid which encodes for a modified channel, hemi-channel, or pore-forming protein in presence of at least one vesicle mimicking a cell membrane, wherein said modified channel, hemi-channel, or pore-forming protein is capable of insertion into the vesicle once it is expressed. Methods for making synthetic cells are known in the art (Shin 2022, for example, herein incorporated by reference in its entirety for its discussion concerning making cells). Emulsion-based methods, such as inverted emulsion and continuous droplet interface crossing encapsulation (cDICE), are best suited for creatingvesicles that incorporate pores or channels. Other methods such as thin-film hydration, gel-assisted swelling, and microfluidics can also be used for this endeavor.

[0072] The synthetic cells can be modified by causing a structure to be embedded therein. This structure can be a channel, pore, opening, etc. as defined above. The synthetic cell (vesicle) can be modified genetically. It can be modified by cell-free protein synthesis. For example, an in vitro transcription-translation system can be used, such as the PURExpress® system. PURExpress® is a reconstituted protein synthesis system based on the PUREsystem™ (Shimizu et al., 2001) where all necessary components needed for in vitro transcription and translation are purified from E. coli. Cell extracts comprising the structure to be embedded can also be used. These cell extracts can be derived from a variety of organisms, e.g., E. coli, rabbit reticulocytes, wheat germ, insect cells, or human cells, for instance. Purified recombinant elements of the protein transcription-translation machinery, such as those found in the PURExpress® can be further modified to create engineered proteins, for e.g., to produce proteins that comprise non-canonical amino acids (See Khambhati et al. Front Bioeng Biotechnol. 2019 Oct 11;7:248 and Cui et al. Front. Bioeng. Biotechnol. 8:1031, both of which are herein incorporated in their entirety).EXAMPLESExample 1: A Fluorescence Assay for Measuring Membrane Channel Activity

[0073] Here, we have successfully developed a new assay to test HC activity in vitro. The method is based on successful reconstitution of connexin 43 (Cx43, a connexin isoform) HCs in micron-sized lipid vesicles, also referred to as synthetic cells. To do this, we encapsulated an in vitro transcription-translation system (PURExpress®) along with plasmid DNA encoding for Cx43-GFP inside vesicles. By incubating these vesicles at 37°C for 2 hours, Cx43-GFP was successfully expressed inside these vesicles. We discovered that Cx43 spontaneously inserts into vesicle membranes and forms functional HCs. Here, we take advantage of Cx43-containing vesicles to assay for HC activity using fluorescence. In order to this, we loaded Cx43-expressing vesicles with a dibenzocyclooctyne (DBCO)-modified casein. We then introduced a membrane -impermeable fluorogenic probe, CalFluor 647, to the external solution that fluoresces upon reaction with DBCO through a Cu-free click reaction. Only in vesicles with active HCs does the fluorogenic probe cross the membrane and react with lumenal DBCO to produce a fluorescence output. This fluorescence can then be read using a plate reader. Indeed, we find a marked fluorescence increase (~20-fold) in vesicles expressing Cx43 overthose that do not. Upon repeating this experiment in the presence of different known HC inhibitors (Gap26 peptide, Ca2+ions, and the small molecule carbenoxolone), we observed a significant decrease in CalFluor 647 fluorescence, indicative of effective HC inhibition (Figure 5). In sum, we have developed an assay based on vesicles / synthetic cells and fluorescence that effectively measures membrane channel activity, opening the door to discovery of new hemichannel inhibitors.Example 2: Cx43-Expressing Vesicles Loaded with DBCO-Sulfo-NHS Ester

[0074] In this version of the assay, instead of loading Cx43-expressing vesicles with DBCO-modified casein, they are loaded with DBCO-sulfo-NHS ester. The sulfo-NHS group makes the DBCO membrane-impermeable. Some amount of it also reacts with the PURExpress components inside our vesicles, further ensuring that it remains sequestered inside the vesicles. Then when CalFluor 488 is added to the vesicle outer solution (Figure 6), it permeates through the Cx43 pores to react with the sulfo-NHS-DBCO and produce fluorescence. The sulfo-NHS-DBCO is amenable to scaling up, such as for conducting inhibitor screens, compared to the DBCO-modified casein.REFERENCES1. Evans WH, Martin PE. Gap junctions: structure and function (Review). Mol Membr Biol. 2002 Apr-Jun; 19(2): 121-36. doi: 10.1080 / 09687680210139839. PMID: 12126230.2. Peng B, Xu C, Wang S, Zhang Y, Li W. The Role of Connexin Hemichannels in Inflammatory Diseases. Biology (Basel). 2022 Feb 2; 11(2):237. doi:10.3390 / biology 11020237.3. Campbell RM. The 2020 SLAS Discovery Top 10: Advancing the Science of Drug Discovery. SLAS Discov. 2021 Feb;26(2): 163-164.4. Krishnan, Srinivasan et al. An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels. SLAS Discovery, Volume 22, Issue 2, 135 - 143.5. Syrjanen J, Michalski K, Kawate T, Furukawa H. On the molecular nature of large-pore channels. J Mol Biol. 2021 Aug 20;433(17): 166994. doi:10.1016 / j.jmb.2021.166994.6. Yeager M. Structure of cardiac gap junction intercellular channels. J Struct Biol.1998;121(2):231-45. doi: 10.1006 / jsbi.1998.3972. PMID: 9615440.7. Lu Y, Allegri G, Huskens J. Vesicle-based artificial cells: materials, construction methods and applications. Mater Horiz. 2022 Mar 7;9(3):892-907.8. Dbouk, H. A., Mroue, R. M., El-Sabban, M. E. et al. Connexins: a myriad of functions extending beyond assembly of gap junction channels. Cell Commun Signal 7, 4 (2009).9. Unger, et al. (1999) Electron cryo-crystallography of a recombinant cardiac gap junction channel, Novartis Found Symp 219: 22-43.10. Xu C, Hu S, Chen X. Artificial cells: from basic science to applications. Mater Today (Kidlington). 2016 Nov;19(9):516-532. doi: 10.1016 / j.mattod.2016.02.020. PMID: 28077925; PMCID: PMC5222523.11. Jooyong Shin, Biair D. Cole, Ting Shan, and Yeongseon Jang. Heterogeneous Synthetic Vesicles toward Artificial Cells: Engineering Structure and Composition of Membranes for Multimodal Functionalities. Biomacromolecules 2022, 23 (4), 1505-1518.12. Suzuki M, Kamiya K. Cell-sized asymmetric phospholipid-amphiphilic protein vesicles with growth, fission, and molecule transportation. ChemRxiv. 2022.Jensen EC. Use of fluorescent probes: their effect on cell biology and limitations. Anal Rec (Hoboken). 2012 Dec;295(12):2031-6. doi: 10.1002 / ar.22602. Epub 2012 Oct 12. PMID: 23060362.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a synthetic cell system, wherein said system comprises:a. at least one vesicle mimicking a cell membrane;b. at least one structure embedded within the vesicle, wherein the structure comprises an opening or channel, wherein the opening or channel can allow' material to pass from one side of the membrane to another; and c. at least one detectable probe.

2. The composition of claim 1, wherein the vesicle comprises lipid material, polymeric material, extracted native cellular material, or a hybrid of any of these.

3. The composition of claim 2, wherein the lipid vesicle comprises a phospholipid bilayer.

4. The composition of any one of claims 1-3, wherein the vesicle further comprises additional components.

5. The composition of any one of claims 1-4, wherein the opening or channel can be open, closed, or semi-open.

6. The composition of any one of claims 1-5, wherein the structure comprises a protein.

7. The composition of claim 5 or 6, wherein the opening or channel can comprise a hemichannel or pore-forming protein.

8. The composition of any one of claims 1-7, wherein the structure comprises connexin, innexin, pannexin, leucine-rich repeat-containing 8 (LRRC8), calcium homeostasis, modulator (CALHM), porin, voltage-gated ion channel, ligand-gated ion channel, ionotropic glutamate receptors, P2X receptors, mechano-sensitive ion channels, or engineered or non-naturally occurring pore-forming proteins.

9. The composition of claim 8, wherein the connexin is CX43 or CX32.

10. The composition of any one of claims 1-9, wherein the probe is fluorogenic.

11. The composition of claim 10, wherein the fluorogenic probe comprises CalFluor 647 or CalFluor 488.

12. The composition of any one of claims 1-11, wherein the composition further comprises a substance which can react with the probe, and further wherein the substance is on an opposite side of the vesicle than the probe.

13. The composition of claim 12, wherein the substance comprises dibenzocyclooctyne (DBCO).

14. The composition of claim 13, wherein the vesicle is loaded with DBCO-modified casein or DBCO-sulfo-NHS ester.

15. The composition of any of claims 12-14, wherein the substance reacts with the probe in a click chemistry reaction.

16. The composition of any one of claims 12-15, wherein, upon reaction of the probe and the substance that reacts with the probe, a detectable signal is produced.

17. The composition of any one of claims 1-16, wherein the composition comprises more than one vesicle, more than one channel or pore forming protein, and / or more than one probe.

18. A method of determining that an analyte affects transport of a molecule across a pore / channel, the method comprising:a. providing a synthetic cell system comprising:i. at least one vesicle mimicking a cell membrane,ii. at least one structure embedded within the vesicle, wherein the structure comprises an opening or channel, wherein the opening or channel can allow material to pass from one side of the membrane to another, iii. at least one detectable probe, andiv. a substance which can react with the probe on an opposite side of the vesicle than the detectable probe;b. exposing the analyte to the synthetic cell system; andc. measuring a change in signal from the detectable probe, wherein a difference in signal indicates that the analyte affects transport of the molecule.

19. A method of making a synthetic cell system with a pore or channel therein, the method comprising providing a plasmid which encodes for a modified channel, hemi-channel, or pore-forming protein in presence of at least one vesicle mimicking a cell membrane, wherein said modified channel, hemi-channel, or pore-forming protein is capable of insertion into the vesicle once it is expressed.

20. The method of claim 19, wherein an in vitro transcription-translation system is used.

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