Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4′), 436 (0′), and 440 (4′) residues by 5-HT3B (-4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β2 nAChRs (γ = 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family. Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4′), 436 (0′), and 440 (4′) residues by 5-HT3B (-4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β2 nAChRs (γ = 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family. There are five families of vertebrate Cys loop receptors as follows: the nicotinic acetylcholine receptor (nAChR), 3The abbreviations used are: nAChR, nicotinic acetylcholine receptors; 5-HT, 5-hydroxytryptamine; HEK, human embryonic kidney cells; MA, membrane-associated; WT, wild type; ANOVA, analysis of variance; 5-HT3AR, 5-hydroxytryptamine type 3A receptor; ACh, acetylcholine; GFP, green fluorescent protein. the 5-hydroxytryptamine type 3 receptor (5-HT3R), the zinc-activated ion channel, the γ-aminobutyric acid type A receptor, and the strychnine-sensitive glycine receptor (1Alexander S.P.H. Mathie A. Peters J.A. Br. J. Pharmacol. 2004; 141: S1-S126Crossref PubMed Scopus (3) Google Scholar, 2Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (167) Google Scholar). Structural analysis by cryo-EM of tubular crystals prepared from the Torpedo marmorata electric organ revealed that five subunits combine in nAChRs, forming a rosette around the central ion channel (3Unwin N. FEBS Lett. 2003; 555: 91-95Crossref PubMed Scopus (138) Google Scholar). The second transmembrane (M2) domain of each subunit participates in lining the channel pore, and collectively they present a hydrophobic constriction adjacent to what is traditionally believed to be the rate-limiting portion of the ion conduction pathway that controls single channel conductance (γ) and ionic selectivity (2Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (167) Google Scholar, 4Peters J.A. Hales T.G. Lambert J.J. Trends Pharmacol. Sci. 2005; 26: 587-594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The homomeric 5-HT3A receptor is unique among Cys loop receptors, having a γ below the resolution of single channel recording, estimated by variance analysis to be in the femtosiemen range. The incorporation of the 5-HT3B subunit into human heteromeric 5-HT3A/B receptors increases γ to 16 pS, enabling direct observation of events by single channel recording from outside-out patches (5Davies P.A. Pistis M. Hanna M.C. Peters J.A. Lambert J.J. Hales T.G. Kirkness E.F. Nature. 1999; 397: 359-363Crossref PubMed Scopus (495) Google Scholar). The use of chimeric 5-HT3A-5-HT3B constructs and site-directed mutagenesis revealed a critical role of three arginine residues within the MA helix of the M3-M4 cytoplasmic loop in determining γ (6Finer-Moore J. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 155-159Crossref PubMed Scopus (383) Google Scholar, 7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) Google Scholar). This has prompted speculation that the MA helix may participate in the control of γ in other Cys loop receptors (4Peters J.A. Hales T.G. Lambert J.J. Trends Pharmacol. Sci. 2005; 26: 587-594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) 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 (363) Google Scholar). In support of this interpretation, cryo-EM analysis revealed portals within the Torpedo nAChR formed, in part, by the MA helices of adjacent subunits that may participate in the ion conduction pathway (9Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (430) Google Scholar). In this study we investigated the influence of the 5-HT3A subunit's MA helix Arg-432 (-4′), Arg-436 (0′), and Arg-440 (4′) residues in the control of γ. We investigated the effect of introducing arginine into the equivalent locations within the nAChR α4 and β2 subunits. Our data confirm the critical role of MA -4′, 0′, and 4′ residues in controlling γ of 5-HT3A receptors and support the hypothesis that the MA helix also forms part of the ion conduction pathway of nAChRs. Our functional data provide support for the existence of cytoplasmic portals depicted in the 4 Å structural model of the nAChR (10Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1442) Google Scholar). DNA Constructs and Transient Transfection of Subunit cDNAs—cDNAs encoding rat wild-type (WT) nAChR α4 and β2 subunits (Dr. J. M. Boulter, Department of Psychiatry and Biobehavioral Sciences, UCLA), human WT 5-HT3A subunits, and mutant nAChR and 5-HT3A subunits were cloned into pGW1 (11Boyd G.W. Low P. Dunlop J.I. Robertson L.A. Vardy A. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (69) Google Scholar). Point mutations were introduced using standard molecular biological techniques (7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) Google Scholar). All cDNAs were sequenced to confirm fidelity. Transfection of tsA-201, or HEK-293 cells, with subunit cDNAs, at equimolar ratios when appropriate, was performed by either the calcium phosphate precipitation method or electroporation (400 V, infinite resistance, 125 microfarads) using a Bio-Rad gene electropulser II. Transfected cells were routinely cultured at 37 °C for 24-72 h before use, although in the case of nicotinic receptor constructs incubation temperature was reduced to 29 °C overnight prior to recording in order to enhance cell surface expression (12Cooper S.T. Harkness P.C. Baker E.R. Millar N.S. J. Biol. Chem. 1999; 274: 27145-27152Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Electrophysiology—Whole-cell and outside-out patch configurations were used to record macroscopic and single channel currents, respectively, from transfected cells. The recording chamber was perfused (5 ml min-1) with a solution of (in mm) NaCl 140, KCl 2.8, MgCl2 2.0, CaCl2 1.0, glucose 10, and macroscopic current (in mm) KCl 140, MgCl2 2.0, CaCl2 mm) was by to cells to of were to cell in order to current and currents, for were by to cells channel were estimated from the macroscopic current to as Lambert J.J. Peters J.A. J. Scopus Google Scholar). analysis was performed using the data (Dr. J. of single channel recordings from outside-out or variance analysis from the were with (in mm) NaCl MgCl2 channel from outside-out patches were either at in receptor experiments or in receptor experiments at in both were as P.A. Kirkness E.F. Hales T.G. J. Scopus Google Scholar). were by to patches at either in the case of WT and mutant 5-HT3A constructs or in the case of nAChR constructs for of data in events were for analysis and were for the of using were to using the method within The of the single channel current from each patch was from the the current from the to the and channel are as the conductance as γ = is current is the and is the of the macroscopic current In outside-out patch recordings from and receptors, were Our analysis of single was to the in to the events in are as ± were using analysis of variance with the the 5-HT3A and nAChR α4 and β2 the 4 Å of the Torpedo acid of 5-HT3A and and WT α4 and β2 subunits were against Torpedo and γ subunits using in loop introduced into the against the were also introduced in the locations of each The 5-HT3A residues in nAChR subunits, and were with residues from the equivalent The of the of from residues were either introduced from the subunit or from the Homology Torpedo was from the into the of 5-HT3A and and WT nAChR α4 and β2 subunits were the Torpedo in The nAChR subunits were the with the in the The models were to for models were to for and residues were from for The subunit the model to the Torpedo (10Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1442) Google Scholar). in the MA of the 5-HT3A Subunit of to outside-out patches from cells transfected with encoding the human 5-HT3A subunit resolvable single channel events with (5Davies P.A. Pistis M. Hanna M.C. Peters J.A. Lambert J.J. Hales T.G. Kirkness E.F. Nature. 1999; 397: 359-363Crossref PubMed Scopus (495) Google Scholar). analysis of by WT 5-HT3A receptors to have a single channel conductance (γ) of ± 75 influence of acid the γ for 5-HT3A and α4β2 constructs The γ were from the of single in recordings from outside-out patches by or analysis was used to γ when resolvable were in outside-out patch was by with and were made WT 5-HT3A and WT α4β2 constructs and within the ± γ was estimated by variance analysis of from either cells or outside-out ± γ was estimated by variance analysis of from either cells or outside-out ± ± ± ± γ was estimated by variance analysis of from either cells or outside-out ± ± ± ± 0′Asp, ± ± ± ± γ was estimated by variance analysis of from either cells or outside-out patches α4β2 ± β2 ± WT β2 ± β2 ± 0′Arg β2 ± WT β2 ± 0′Arg β2 ± was by with and were made WT 5-HT3A and WT α4β2 constructs and within the This γ was estimated by variance analysis of from either cells or outside-out (7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) Google in a We the human 5-HT3A subunit acid with of the human 5-HT3B subunit and nAChR subunits, the marmorata and subunits. The 5-HT3A was to the 4 Å structural model of the Torpedo nAChR (10Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1442) Google and the model was The 5-HT3A subunit a of arginine residues within the MA helix we residues MA -4′, 0′, and 4′ for of with the residues in the nAChR subunits By using variance analysis of currents, we that arginine residues at MA -4′ and 4′ participate with MA 0′ in the control of γ. In single channel recordings of from outside-out patches mutant homomeric receptors (in MA -4′, 0′, and 4′ residues were by 5-HT3B residues to as the revealed current events with a γ of 36.5 ± 1.0 and in γ in to WT 5-HT3A The of γ by variance analysis is and in of this the of γ from direct observation of single channel events by the receptor is that we using a solution (7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) Google is in with by using a recording M. M. J. Neurosci. 2005; PubMed Scopus Google Scholar). we the contribution of the MA -4′, 0′, and 4′ residues in the control of γ and in by single channel resolution the effect of the MA -4′, 0′, and 4′ residues in 5-HT3A with equivalent residues from the 5-HT3B of γ using single channel analysis are to of γ that we using variance There is a γ using = The of γ using variance analysis be by the of events below the resolution of single channel recording or the use of recording of to outside-out patches from cells the receptor produced current channel events were analysis of current from outside-out patches a conductance to that (7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) Google using analysis of variance for WT 5-HT3A and mutant This that of γ using variance analysis are influenced by the or outside-out patch recording A role for the -4′ arginine in the control of γ be by the of by the in the -4′ arginine is to the of by in the was by patches receptors single channel events of a reduced γ in to receptors data Arg-432 to have a influence γ. In to the replacement of Arg-436 of the 5-HT3A subunit by the equivalent 5-HT3B subunit residue a substantial in γ that single channel events were in outside-out patch recordings the we γ for by the mutant receptor to be ± The of Arg-436 is by the receptor, single of a reduced conductance with the receptor The of the 5-HT3A Arg-440 residue by the equivalent 5-HT3B residue produced in γ with the WT 5-HT3A receptor the γ of the receptor is reduced to the receptor the data that for the 5-HT3A receptor the of the residue the MA 0′ is determinant of Arg-440 and to a Arg-432 are also in this of MA 0′ of nAChR into the 5-HT3A Subunit γ of nicotinic receptors and that of the 5-HT3A that the γ of the heteromeric receptor the homomeric 5-HT3A receptor is at in part, to the of MA residues and the MA 0′ residue in we investigated the influence of the residues that MA 0′ in the nAChR and subunits the γ of the 5-HT3A receptor of the receptor single channel events that were in outside-out patch recordings the γ of the receptor was to that of the receptor, that MA 0′ residue in the and MA 0′ residue in the 5-HT3B residues are in γ at this The receptor also single The γ of the receptor was reduced in to the receptor for the 5-HT3A receptor, constructs that MA 0′ residue of the β2 although is as in this as or single channel events were resolvable in outside-out patch recordings of receptors, although current were present analysis of that this receptor has a γ below the resolution of single channel recording the receptor a γ estimated by analysis of the patch current experiments that the into the 5-HT3A subunit of the MA 0′ residues in the nAChR α4 subunits substantially increases γ. for a of events in the effect of the 5-HT3A mutations we to the variance of single channel with γ. There was the channel current variance and γ of mutant and receptors = from data A of channel current variance and γ that in γ are of Replacement of MA 4′ of α4β2 nAChRs by to the WT homomeric 5-HT3A receptor, single channel events by both homomeric and heteromeric nAChRs have γ in the picosiemen range (12Cooper S.T. Harkness P.C. Baker E.R. Millar N.S. J. Biol. Chem. 1999; 274: 27145-27152Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, M. S.P. J. Neurosci. PubMed Google Scholar, A. M. J. J. Neurosci. PubMed Google Scholar, S. A. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, of Scholar). patch recordings from that the nAChR has a γ of S. A. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the influence of the nicotinic MA -4′, 0′, and 4′ residues the γ of the 5-HT3A receptor, we the of the equivalent residues in nAChR subunits to the γ of the homomeric receptors in cells; we to study α4β2 This receptor the that MA residues be introduced into or of the subunits within heteromeric nAChRs by mutant α4 and β2 subunits or in with WT subunits. We transfected cells with mutant and constructs the and 4′ residues of both subunits to the residue in the 5-HT3A to single in outside-out patches from transfected cells patches By from patches WT α4β2 receptors and the of mm) to cells in of the 16 cells although cells of cells of cells transfected with WT α4β2 cDNAs current By the of current to cell we the current density by WT α4β2 receptors mm) was also cells WT α4 and mutant β2 or we investigated the influence the current to (1Alexander S.P.H. Mathie A. Peters J.A. Br. J. Pharmacol. 2004; 141: S1-S126Crossref PubMed Scopus (3) Google of MA -4′, 0′, and 4′ mutations within or nAChR subunits. were for the -4′ and 0′ either with the mutant or a WT cells was for the receptor when the 4′ was by either the α4 or the the current was reduced single were from outside-out patches from cells. the of the glutamate residue by the arginine residue at the 4′ of either the α4 or the β2 subunit has a effect the functional expression of the We are the of this and we that in both the α4 and β2 subunits the a for by of the MA 4′ α4 β2 residues by arginine current density by by mm) to cells WT or the mutant α4β2 nAChR at the of the The the current from cells WT and mutant α4 and β2 subunit were by the current by the of each mutant subunit that a in current by with Replacement of MA -4′ or 0′ of α4β2 nAChRs by to the MA 4′ resolvable single be for receptors the -4′ or 0′ We the role of the MA 0′ residue in In to WT α4β2 nAChRs, the γ of receptors from and subunits was when both by analysis of A and and by direct observation of single channel events and when the mutant was with WT β2 subunits, was a of γ from outside-out patch recordings and when the mutant was with the WT α4 γ was reduced and influence of γ. channel channel in to of Scholar). the that a for the in γ by of MA 0′ residues in α4 and β2 subunits, we to the channel current variance from the to to the of WT and mutant α4β2 receptors channel the in γ and variance is a to the data of γ current variance revealed a of of A of that in γ be by a to events of Scholar). data that the of arginine residues into the MA 0′ of α4 and β2 subunits a in γ. We the contribution of the MA residue to the γ of α4β2 nAChRs. The -4′ a effect the single channel conductance of 5-HT3A receptors when with that of the 0′ residue and of arginine at this in both the α4 and β2 subunits a and may be to have a effect conductance in this the γ of receptors from and subunits was substantially reduced (γ = ± 0.5 A and when with that of WT α4β2 nAChRs In common with the nAChR 0′ when the -4′ was introduced into of the subunits, either the α4 or the β2 was a of γ. In the α4β2 receptor the γ of of the nAChRs a single of subunits (γ = ± that in common with the 5-HT3A receptor, the of the MA 0′ residue the γ of the α4β2 for the 5-HT3A receptor, the of the -4′ residue γ was in to that of the 0′ and for the α4β2 nAChR the influence of the -4′ residue is that of the 0′ for the α4β2 nAChR the -4′ or the 0′ arginine residue be introduced both the α4 and the β2 subunit to the of γ. This study that of the cytoplasmic loop within the to as either the MA helix (10Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1442) Google or (6Finer-Moore J. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 155-159Crossref PubMed Scopus (383) Google influence the single channel conductance both the and receptors; a traditionally to the (2Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (167) Google Scholar, 4Peters J.A. Hales T.G. Lambert J.J. Trends Pharmacol. Sci. 2005; 26: 587-594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A is from of the Torpedo nAChR of to MA into the to that the of the ion channel (9Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (430) Google Scholar, N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1442) Google Scholar). of the the of the nAChR that adjacent subunits. The of the portals are estimated to be with of the and be to influence the of ion (9Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (430) Google Scholar, N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1442) Google Scholar). we that within the MA helices of the homomeric 5-HT3A receptor five portals In each three arginine residues -4′, 0′, and a rate-limiting to ion conduction (7Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (236) Google Scholar). residues are for the femtosiemen conductance that is a unique of homomeric 5-HT3A receptors (5Davies P.A. Pistis M. Hanna M.C. Peters J.A. Lambert J.J. Hales T.G. Kirkness E.F. Nature. 1999; 397: 359-363Crossref PubMed Scopus (495) Google Scholar). In contrast, the 5-HT3B subunit homomeric receptors increases the γ of heteromeric 5-HT3A/B receptors to pS. We produced models of the 5-HT3A and α4β2 receptors by acid the 4 Å resolution of the Torpedo The homomeric 5-HT3A receptor model is by of the that the were from a heteromeric this that MA -4′, 0′, and 4′ arginine residues are at the cytoplasmic of the 5-HT3A portals of 5-HT3B subunit MA 0′Asp, and residues into the model that residues a to ion a for the γ in recordings from the receptors The mutant receptor has a γ with that of the WT 5-HT3A is that the density of residues in also participates in γ. is that are residues lining the α4β2 nAChR portals of the residues at MA -4′, 0′, and 4′ locations within the 5-HT3A α4 and β2 subunits have residues at both the MA -4′ and 4′ The portals of the nAChRs also by residues lining cytoplasmic with of the 5-HT3A receptor are in with the substantially γ for α4β2 nAChRs with 5-HT3A The MA 0′ residue has the γ of single 5-HT3A receptor residue Replacement of the MA 0′ arginine residue of the 5-HT3A subunit by the equivalent residue in the 5-HT3B subunit is to γ from the resolution of direct to enabling direct observation of events in outside-out patch of MA 0′ residues of either or β2 subunits and into the 5-HT3A receptor also substantially γ. of the MA 0′ arginine by the MA 0′ residue of the α4 by contrast, reduced the γ of phenylalanine is a and to γ within the femtosiemen range the hypothesis that the of the MA 0′ residue is determinant of γ. The critical role of the 5-HT3A MA 0′ residue in controlling γ is by the 0′ with arginine in the γ This to in γ The MA -4′ and 4′ residues 0′ in the MA helix also a contribution to γ. This be most when either the -4′ or the 4′ are to the the single channel of and are reduced by and respectively, with of arginine into either the MA -4′ or 0′ locations of the α4β2 nAChR also in γ. This effect was most when arginine was introduced into either the -4′ or 0′ locations of both the α4 and β2 subunits and receptors, when the γ of the mutant receptors were reduced by and respectively, from that of the WT five portals of each of the mutant receptors residues at critical in the cytoplasmic conduction the of into the MA -4′ and 0′ locations of the α4 and β2 subunits substantially reduced mutant nAChRs that of the WT 5-HT3A is that the density of residues in the 5-HT3A receptor is to the femtosiemen γ. this we mutant α4 and β2 in with WT α4 and β2 or mutant constructs to functional receptor expression to of γ. of arginine into the MA 4′ of either the α4 or β2 subunits a of functional We that this a experiments be to the of the reduced current A of the the of MA -4′, 0′, and 4′ residues and γ of and receptors is to in ion this study that the of the MA -4′ and 0′ residues in both α4 and β2 subunits to a substantial of that cytoplasmic residues influence ion conduction heteromeric α4β2 nAChRs in a to that in homomeric 5-HT3A data support a role for cytoplasmic portals in the ion conduction of Cys loop We Unwin for and critical this