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• W2014242135 abstract "To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClβ receptor of Caenorhabditis elegans and the vertebrate cationic α7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClβ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClβ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the PCl/PNa ratios, but the GluClβ receptor's mutants retained high capacity to select Cl- over Na+. These results indicate that, unlike the proposed case of anionic Gly- and γ-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClβ receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClβ receptor's channel, Cl- interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore. To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClβ receptor of Caenorhabditis elegans and the vertebrate cationic α7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClβ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClβ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the PCl/PNa ratios, but the GluClβ receptor's mutants retained high capacity to select Cl- over Na+. These results indicate that, unlike the proposed case of anionic Gly- and γ-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClβ receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClβ receptor's channel, Cl- interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore. The invertebrate GluCl 2The abbreviations used are: GluCl, glutamate-gated chloride; GluClβR, GluClβ receptor; GABA, γ-aminobutyric acid; ACh, acetylcholine; AChR, ACh receptor; 5HT, 5-hydroxtryptamine; 5HT3AR, serotonin receptor; Erev, reversal potential. receptor channels are pentameric transmembrane receptors belonging to a wide superfamily of Cys-loop receptors activated by various neurotransmitters such as acetylcholine (ACh), serotonin (5-hydroxtryptamine, 5HT), γ-aminobutyric acid (GABA), Gly, Glu, or histamine (Fig. 1A) (1Changeux J.-P. FIDIA Res. Found. Neurosci. Award Lec. 1990; 4: 21-168Google Scholar, 2Smith G.B. Olsen R.W. Trends Pharmacol. Sci. 1995; 16: 162-168Abstract Full Text PDF PubMed Scopus (452) Google Scholar, 3Bargmann C.I. Science. 1998; 282: 2028-2033Crossref PubMed Scopus (729) Google Scholar, 4Taylor P. Osaka H. Molles B. Keller S.H. Malany S. Neuronal Nicotinic Receptors (Handbook of Experimental Pharmacoloy). 144. Springer Verlag, Berlin2000: 79-100Google Scholar, 5Karlin A. Nat. Rev. Neurosci. 2002; 3: 102-114Crossref PubMed Scopus (781) Google Scholar, 6Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 7Fuchs S. Kasher R. Balass M. Scherf T. Harel M. Fridkin M. Sussman J.L. Katchalski-Katzir E. Ann. N. Y. Acad. Sci. 2003; 998: 93-100Crossref PubMed Scopus (17) Google Scholar, 8Lester H.A. Dibas M.I. Dahan D.S. Leite J.F. Dougherty D.A. Trends Neurosci. 2004; 27: 329-336Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). This superfamily consists of cationic channels permeable to Na+, K+, and, in many subunit combinations, to Ca2+ ions, as well as of anionic channels selective to Cl- ions (reviewed by Keramidas et al. (9Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (161) Google Scholar)). Structural similarities shared by Cys-loop receptors enabled swapping of pore sequences between cationic and anionic channels so as to assess the involvement of specific amino acids in ion charge selectivity. It was previously shown that concomitant replacement of the residues at positions -2′, -1′, and 13′ (Fig. 1, B and C) of cationic receptors by the residues found at the homologous positions of anionic receptors, and vice versa, lead to conversion of ion charge selectivity (10Galzi J.L. Devillers-Thiery A. Hussy N. Bertrand S. Changeux J.P. Bertrand D. Nature. 1992; 359: 500-505Crossref PubMed Scopus (344) Google Scholar, 11Keramidas A. Moorhouse A.J. French C.R. Schofield P.R. Barry P.H. Biophys. J. 2000; 79: 247-259Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 12Gunthorpe M.J. Lummis S.C. J. Biol. Chem. 2001; 276: 10977-10983Abstract Full Text Full Text PDF Scopus (116) Google Scholar, 13Menard C. Horvitz H.R. Cannon S. J. Biol. Chem. 2005; 280: 27502-27507Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Further mutagenesis studies led to the recognition that the different capacities of cationic versus anionic Cys-loop receptors to distinguish between the charge of ions rely on the differences in the amino acid composition at positions -1′ and -2′ (Fig. 1C) (13Menard C. Horvitz H.R. Cannon S. J. Biol. Chem. 2005; 280: 27502-27507Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Corringer P.J. Bertrand S. Galzi J.L. Devillers-Thiery A. Changeux J.P. Bertrand D. Neuron. 1999; 22: 831-843Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (80) Google Scholar, 16Wotring V.E. Miller T.S. Weiss D.S. J. Physiol. (Lond.). 2003; 548: 527-540Crossref Scopus (52) Google Scholar). The conserved pore-facing Glu residue at position -1′ of cationic Cys-loop receptors was further inferred to form, around the axis of ion conduction, a negatively charged ring that plays the key role in cationic selectivity by interacting with cations and repulsing anions (12Gunthorpe M.J. Lummis S.C. J. Biol. Chem. 2001; 276: 10977-10983Abstract Full Text Full Text PDF Scopus (116) Google Scholar, 13Menard C. Horvitz H.R. Cannon S. J. Biol. Chem. 2005; 280: 27502-27507Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 15Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (80) Google Scholar, 16Wotring V.E. Miller T.S. Weiss D.S. J. Physiol. (Lond.). 2003; 548: 527-540Crossref Scopus (52) Google Scholar). Conversely, a conserved arginine at position 0′ of anionic Cys-loop receptors was inferred to interact with anions and repulse cations. A basic residue at position 0′ is also typical of all cationic Cys-loop receptors (Fig. 1C and the ligand-gated ion channels data base), but it was suggested that local conformational differences in the M1-M2 connecting segment (M1-M2 loop) orient this basic residue to the pore lumen only in anionic Cys-loop receptors (9Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (161) Google Scholar, 15Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (80) Google Scholar). These local conformational differences have been attributed to a proline residue, which is present exclusively at position -2′ of anionic Cys-loop receptors (12Gunthorpe M.J. Lummis S.C. J. Biol. Chem. 2001; 276: 10977-10983Abstract Full Text Full Text PDF Scopus (116) Google Scholar, 14Corringer P.J. Bertrand S. Galzi J.L. Devillers-Thiery A. Changeux J.P. Bertrand D. Neuron. 1999; 22: 831-843Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (80) Google Scholar, 16Wotring V.E. Miller T.S. Weiss D.S. J. Physiol. (Lond.). 2003; 548: 527-540Crossref Scopus (52) Google Scholar, 17Lee D.J. Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Neurosci. Lett. 2003; 351: 196-200Crossref PubMed Scopus (27) Google Scholar). Unlike all other homomeric anionic Cys-loop receptors studied thus far, the β subunit of the GluCl receptor does not have a proline residue at position -2′, a feature that minimizes the likelihood of causing drastic local conformational changes when mutating its M1-M2 loop. As the GluClβ subunit can assemble into a functional anionic homomeric receptor (GluClβR) (18Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H. Schaeffer J.M. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (579) Google Scholar), the selectivity filter of the GluClβR was readily identified here by microchimerism and then was extensively mutated. Electrophysiological analyses of ionic permeability ratios in a large repertoire of mutants, together with computer-assisted molecular modeling, reveal a novel mechanism of Cl- selection by a Cys-loop receptor. Chimeras and Mutants—The α7-GluClβ chimeric subunit was prepared as performed previously with the α7-5HT3AR (19Eisele J.L. Bertrand S. Galzi J.L. Devillers-Thiery A. Changeux J.P. Bertrand D. Nature. 1993; 366: 479-483Crossref PubMed Scopus (361) Google Scholar), by fusing the N-terminal half of the chick α7 subunit (Fig. 1A, red segment) (Swiss-Prot accession number P22770) to the C-terminal half of the β subunit of the GluClR (Fig. 1A, non-red segments) (Swiss-Prot accession number Q17328). The entire sequence of the chimera is provided in Supplemental Fig. S1. Mutations were introduced as described previously (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar). Electrophysiology—Human embryonic kidney (HEK-293) cells were transfected with the various chimeras together with a green fluorescent protein (cloned in a separate plasmid), using the calcium phosphate method. Electrophysiological recordings were made 2-3 days after the transfection, using the whole-cell patch clamp technique. To determine ionic permeability ratios, current-voltage relations were established in various external solutions with and without 100 μm ACh as follows. Solution A contained: 140 mm NaCl, 2.8 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm glucose, 10 mm HEPES, and 5 mm NaOH, pH 7.35. Solutions B1,2... are as solution A, but NaCl was diluted with a mannitol-containing solution to maintain equiosmolar conditions while reducing the external NaCl concentrations to (in mm): B1 = 70, B2 = 35, B3 = 17.5, B4 = 0. Note that in one exceptional case (chimera 4), the composition of the diluted solutions was slightly different, giving external NaCl concentrations of (in mm): B1′ = 100, B2′ = 40, B3′ = 20, B4′ = 10, B5 = 0. Solutions C1,2... are as solution A, but NaCl was replaced by sodium isethionate so as to keep the external concentration of Na+ ions constant and to replace Cl- ions by isethionate. The external concentrations of NaCl in these solutions was (in mm): C1 = 70, C2 = 35, C3 = 17.5, C4 = 0. Solution D is as solution A, but NaCl was replaced by 140 mm sodium acetate. Patch pipettes (1-2 megaohms) were filled with a solution containing 130 mm CsCl, 4 mm MgCl2, 4 mm Na2-ATP, 1 mm EGTA, 10 mm HEPES, and 10 mm CsOH, pH 7.35. External solutions were delivered by a computer-driven valve manifold system enabling fast exchange of the solutions. Current-voltage relations were determined at 25 °C by two methods. (i) Currents evoked by 100 μm ACh for 3 s were measured at different holding potentials ranging from -100 to +50 mV, and (ii) inverted 200-ms long voltage ramps (either from +50 to -100 mV or from +70 to -100 mV) were applied 1 and 2 s after the beginning of a 3-s ACh application. The initial holding potential was -60 mV. Leak currents obtained by the same protocol, in the absence of ACh, were subtracted. Measured reversal potential (Erev) values were corrected to account for the liquid junction potentials by using the JPCalc software (21Barry P.H. J. Neurosci. Methods. 1994; 51: 107-116Crossref PubMed Scopus (547) Google Scholar) implemented in pClamp version 8.1. Permeability ratios for Na+, Cs+, and Cl- were calculated using the Goldman-Hodgkin-Katz equation, Erev=RTFln[Cl−]in+α[Na+]out+β[Cs+]out[Cl−]out+α[Na+]in+β[Cs+]in where R, T, and F are the gas constant, the absolute temperature, and the Faraday's constant, respectively, and the permeability ratios are α = PNa/PCl and β = PCs/PCl. Note that ion activities, instead of ion concentrations, have been used in the analyses of permeability ratios. Ionic activities were calculated on the basis of the Debye-Hückel theory (22MacInnes D.A. The Principles of Electrochemistry. Dover Publications, Inc., New York1961Google Scholar) but with the corrections introduced in the Millero-Pitzer method for solutions having an ionic strength greater than 0.1 m (41Pitzer K.S. Activity Coefficients in Electrolyte Solutions, 2nd Ed. CRC Press, Boca Raton, FL1991: 75-153Google Scholar), as implemented in the Electrolytes program of Aq-Solutions, a software package of programs for the quantitative treatment of equilibria in solution. 3For further information about this software package, please contact the author (Y. P.). Model Building—An initial homology model was built by using the atomic coordinates of the AChR structure (23Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1081) Google Scholar) as a template (Protein Data Bank number 1OED), as recently described (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar). After modeling the M1-M2 loop, it was tilted together with the M1 and M2 segments to an intermediate position between the closed and open states previously elaborated by Paas et al. (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar) in the case of another Cys-loop chimeric receptor. We readily obtained an intermediate position displaying a distance of ∼6.1 Å between the van der Waals surfaces of backbone amides located on opposite sides of the pore, at the level of position -3′. This distance is within the range of the effective open pore diameter determined here. Note that the M1-M2 loop of the GluClβR (DLHSTAG) is shorter by two amino acids than the loop of the models elaborated in Paas et al. (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar) (PPDLHSTAG). As a result, when compared with its position in the chimera modeled in Paas et al. (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar), histidine(H)-5′ of the GluClβR's pore (modeled here) moved away from the permeation pathway. Consequently, H-5′ of the GluClβR's model is not in contact with the permeating ions. This modeling observation is in line with the incapacity of Zn2+ to block the chimeric α7-GluClβR (data not shown), unlike the case of a chimera having the transmembrane segments of the 5HT3AR with the sequence PPDLHSTAG between M1 and M2 (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar). The model of chimera 17 was built as above but with a proline, instead of alanine, at position -2′. The root mean square difference between the backbone atoms of the GluClβR and chimera 17 is 0.03 Å (throughout the pentameric membrane-embedded domains). The root mean square difference between the backbone atoms of the M1-M2 loops (plus their flanking residues, I-10′ and R0′) of these structures is 0.08 Å. Chimeric Design and Current Amplitudes of the Various Mutants—As a first step, we generated a chimeric subunit where the extracellular segment of the AChR α7 subunit was fused to the GluClβ subunit segment that folds in the membrane and cytoplasm (see “Experimental Procedures” and supplemental Fig. S1). The α7-GluClβ chimeric subunit assembles into a homopentameric receptor (α7-GluClβR), like the case of all the mutants studied here. As such, the resulting α7-GluClβR responds to ACh but not to glutamate, allowing us to express our chimeric mutants in HEK-293 cells grown in serum-containing medium with no need of glutamate depletion. That is, this chimeric receptor carries the entire pore of the weakly desensitizing GluClβR (18Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H. Schaeffer J.M. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (579) Google Scholar) without being constitutively open (by glutamate present in the medium) and cytotoxic to the cells. Fig. 2A shows typical current traces of the chimeric α7-GluClβR measured at different membrane voltages. Then, based on previous studies carried out on a few vertebrate Cys-loop receptors, mutations were introduced in regions suspected to contribute to ion charge selectivity. This approach resulted in chimeric mutants that provided robust responses (Table 1).TABLE 1Permeability ratios (PX/PY) determined for the chimeric α7-GluClβR and its mutants Note that: (i) data are means ± S.E.; (ii) all values were rounded to the closest decimal number; (iii) pair sequence alignment between the α7 and the GluClβ receptors dictates the movement of the α7G-3′ (alignment in Fig. 1C) by one position downstream, i.e., it is G-2′ in Table I; (iv) gaps in the sequence are also numbered; (v) residues shown in blue do not appear in the native sequence of either the α7-AChR or the GluClβR; (vi) the entire sequence of the α7-GluClβ chimeric subunit is provided in the Supplemental Data (Fig. S1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)a Measured in response to 100 μm acetylcholine at -60mVb Number of cellsc Ion charge selectivity: A, anionic; C, cationicd The PCl/PCs ratio of chimeras 2 or 12 does not statistically differ from that of the α7-GluClβR (P = 0.059 and 0.150, respectively; tow-tailed, unpaired t test)e The PCl/PCs ratio of chimeras 13, 14 or 15 significantly differs from that of the α7-GluClβR (P = 0.0015, 0.0025, and 0.0009, respectively; two-tailed, unpaired t test)f NF, not functional Open table in a new tab a Measured in response to 100 μm acetylcholine at -60mV b Number of cells c Ion charge selectivity: A, anionic; C, cationic d The PCl/PCs ratio of chimeras 2 or 12 does not statistically differ from that of the α7-GluClβR (P = 0.059 and 0.150, respectively; tow-tailed, unpaired t test) e The PCl/PCs ratio of chimeras 13, 14 or 15 significantly differs from that of the α7-GluClβR (P = 0.0015, 0.0025, and 0.0009, respectively; two-tailed, unpaired t test) f NF, not functional Ion Charge Selectivity of the α7-GluClβR and Its Various Mutants—The I-V relations plotted for the α7-GluClβR show that omission of almost all external Cl- ions shifts the reversal potential (Erev) to a positive voltage (e.g. Fig. 2B), by 54.5 ± 1.2 mV (mean ± S.E. from 12 cells, after correcting with liquid junction potentials). The latter value is close to the maximal theoretical shift calculated based on the Cl- equilibrium (Nernst) potential (61.4 mV; calculated using ionic activities). The extent of the shift depended on the external concentrations of Cl- (Fig. 3A) and showed that the α7-GluClβR chimera is highly selective to Cl- relative to Na+ and Cs+ (PCl/PNa = ∼45 and PCl/PCs = 26; Table 1), as expected from a chimera harboring the pore of the GluClβR (18Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H. Schaeffer J.M. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (579) Google Scholar). Previous studies suggested that a positively charged residue substituted at the extracellular mouth of Cys-loop receptor mutants (position 19′) partially contributes to ionic selectivity by attracting Cl- ions (24Thompson A.J. Lummis S.C. Br. J. Pharmacol. 2003; 140: 359-365Crossref PubMed Scopus (54) Google Scholar) or repulsing divalent cations (15Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (80) Google Scholar). It was also shown that an Arg19′ → Glu mutation in an anionic-to-cationic converted glycine receptor mutant contributes to cationic conductance (25Moorhouse A.J. Keramidas A. Zaykin A. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 411-425Crossref PubMed Scopus (36) Google Scholar). We therefore replaced the segment 19′NAKL22′ of the GluClβR pore by the homologous segment of α7 (19′AEIM22′), and the resulting chimera retained Cl- selectivity, albeit with a lower PCl/PNa ratio, but with no change in the PCl/PCs ratio (chimera 1, Table 1 and Supplemental Fig. S2A). Cl- selectivity was also observed when the positive charge of the first amino acid of M2 (position 0′) was neutralized (chimera 2, Table 1 and Figs. 2C and 3A). Position -1′ is located in a constriction that extends from position 2′ toward the bottom of the pore (20Paas Y. Gibor G. Grailhe R. Savatier-Duclert N. Dufresne V. Sunesen M. de Carvalho L.P. Changeux J.P. Attali B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15877-15882Crossref PubMed Scopus (65) Google Scholar, 26Imoto K. Busch C. Sakmann B. Mishina M. Konno T. Nakai J. Bujo H. Mori Y. Fukuda K. Numa S. Nature. 1988; 335: 645-648Crossref PubMed Scopus (605) Google Scholar, 27Imoto K. Konno T. Nakai J. Wang F. Mishina M. Numa S. FEBS Lett. 1991; 289: 193-200Crossref PubMed Scopus (102) Google Scholar, 28Villarroel A. Sakmann B. Biophys. J. 1992; 62: 196-208Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 29Bormann J. Rundstrom N. Betz H. Langosch D. EMBO J. 1993; 12: 3729-3737Crossref PubMed Scopus (217) Google Scholar, 30Wilson G.G. Karlin A. Neuron. 1998; 20: 1269-1281Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Consistently, it was previously shown that a substitution of Glu at position 2′ of the homomeric Glyα1R converted its selectivity (31Carland J.E. Moorhouse A.J. Barry P.H. Johnston G.A. Chebib M. J. Biol. Chem. 2004; 279: 54153-54160Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In addition, Glu substituted at positions -3′ or -4′ of the β subunit of a heteromeric GABAAα2β3γ2 receptor imparted permeability to cations along with anions (32Jensen M.L. Pedersen L.N. Timmermann D.B. Schousboe A. Ahring P.K. J. Neurochem. 2005; 92: 962-972Crossref PubMed Scopus (20) Google Scholar). Here, concomitant replacement of the M1-M2 loop and the residues at positions 0′ and 2′ of the GluClβR's pore by those of α7 resulted in a fully cationic channel (chimera 3, Table 1 and supplemental Fig. S2B). Replacing only the M1-M2 loop also produced a fully cationic channel, indicating that a hydroxyl group at position 2′ does not play a role in ion charge selectivity (chimera 4, Table 1 and Figs. 2E and 3B). The latter conclusion well agrees with the observations that following non-polar substitutions at position 2′, the muscle AChR retains permeability to monovalent cations that are considered to interact with this position while traversing the pore (28Villarroel A. Sakmann B. Biophys. J. 1992; 62: 196-208Abstract Full Text PDF PubMed Scopus (63) Google Scholar). Cationic selectivity was also observed when chimera 4 was further modified so as either to carry the M1-M2 sequence of the cationic 5HT3AR (chimera 5, Table 1 and supplemental Fig. S2C) or to carry a neutral residue at position -7′ (chimera 6, Table 1, Fig. 3B, and supplemental Fig. S2D). The latter modification indicates that the so-called cytoplasmic ring (D-7′) does not play a role in ion charge selectivity. As long as a Glu residue occupied position -1′, further gradual changes in the sequence of the M1-M2 loop toward the sequence of the GluClβR (chimeras 7 and 8) did not convert the mutants back to anionic receptors, but they retained cationic selectivity, even when the GluClβR's pore was carrying a single G-1′E substitution (chimera 9) (Table 1, Figs. 2F and 3B, and supplemental Fig. S2E and S2F). It should, however, be noted that in three cases, a slight decrease in the relative permeability to Na+ (i.e. increase in PCl/PNa) was observed (chimeras 6, 7, and 9, Table 1). In contrast, integrating the residues of the M1-M2 loop of α7 within the sequence of the GluClβR's loop while keeping a Gly at position -1′ (i.e. the native amino acid of the GluClβR) provided an anionic channel (chimera 10, Table 1 and supplemental Fig. S2G). Deleting the GluClβR residues at positions -6′ and -5′ (LH) slightly reduced the PCl/PNa and PCl/PCs ratios, but these mutants still displayed considerable capacity to select Cl- over Na+ and Cs+ (chimeras 11 and 12, Table 1, Fig. 3A, and supplemental Fig. S2H and S2I). Further deletion of the Thr that precedes position -2′ in the GluClβR, alone (chimera 13) or together with neutralization of the charge either at position -7′ (chimera 14) or at position 0′ (chimera 15), resulted in channels that retained high Cl- over Na+ selectivity but became slightly permeable to Cs+ (Table 1, Figs. 2D and 3A, and supplemental Fig. S2J and S2K). Shortening the M1-M2 loop to four amino acids rendered the chimeric receptor nonfunctional (chimera 16, Table 1). Proline at position -2 was shown to play a role in determining the (small) pore diameter of a glycine receptor, indicating that pore size also contributes to ion charge selectivity (Ref. 17Lee D.J. Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Neurosci. Lett. 2003; 351: 196-200Crossref PubMed Scopus (27) Google Scholar, reviewed by Keramidas et al. (9Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (161) Google Scholar)). Interestingly, an α7-GluClβR having a proline at position -2′ (chimera 17) displays PCl/PNa and PCl/PCs ratios closely similar to those of the α7-GluClβR (Table 1 and supplemental Fig. S3). Relative Permeability of Chloride-selective Chimeras to Isethionate and Acetate—To assess the dimensions of the open pore of the α7-GluClβR and anionic mutants, we examined the permeability to the organic anions isethionate and acetate relatively to the permeability for Cl-. Table 2 shows that the α7-GluClβR is not permeable to isethionate but allows acetate to permeate slightly. Elimination of the side chain at position 0′ (chimera 2) or deleting residues belonging to the M1-M2 loop of the GluClβR (chimeras 12-15) turned these mutants permeable to isethionate and increased the relative permeability to acetate (Table 2). The increase in the relative permeability to isethionate linearly correlated with the increase in the relative permeability to acetate (Fig. 4), indicating that these mutations have effectively widened the open pore. Notably, the α7-GluClβR-A-2′P mutant (chimera 17) was found to be as permeable to acetate as the α7-GluClβR (PAcet/PCl = 0.1 ± 0.013, mean ± S.E. from 5 cells; e.g. supplemental Fig. S3A).TABLE 2Relative permeability to isethionate and acetate Note: Data are means ± S.E. values. The unhydrated dimensions of the two organic anions, in Å, are: isethionate (Ise), 5.8 × 6.2 × 8.1 and acetate (Acet), 3.99 × 5.18 × 5.47. Note that the 2nd largest anion dimension (bold) was used for estimating the range of the effective open pore diameter.ChimeraPIse/PClNaNumber of cellsPAcet/PClNα7-GluClβR0.00 ± 0.000120.09 ± 0.0107Chimera 120.09 ± 0.01470.22 ± 0.0306Chimera 20.13 ± 0.01080.25 ± 0.0196Chimera 130.16 ± 0.01760.37 ± 0.0205Chimera 150.19 ± 0.01860.46 ± 0.0266Chimera 140.26 ± 0.016110.56 ± 0.0297a Number of cells Open table in a new tab The Location of the Selectivity Filter in the GluClβR's Channel—Previous studies on the homomeric ACh-α7, Glyα1, and 5HT3 receptors had shown that concomitant substitutions at positions -2′, -1′, and 13′ converted their ion charge selectivity (10Galzi J.L. Devillers-Thiery A. Hussy N. Bertrand S. Changeux J.P. Bertrand D. Nature. 1992; 359: 500-505Crossref" @default.
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• W2014242135 title "Mechanism of Cl- Selection by a Glutamate-gated Chloride (GluCl) Receptor Revealed through Mutations in the Selectivity Filter" @default.
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• W2014242135 doi "https://doi.org/10.1074/jbc.m511657200" @default.
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