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• W2035499881 abstract "The determinants of single channel conductance (γ) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that γ is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp113 and Asp127) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT3A) receptor (5-HT3A(QDA)) upon γ, modulation of the latter by extracellular Ca2+, and the permeability of Ca2+ with respect to Cs+ (PCa/PCs). Mutations neutralizing (Asp → Asn), or reversing (Asp → Lys), charge at the 113 locus decreased inward γ by 46 and 58%, respectively, but outward currents were unaffected. The D127N mutation decreased inward γ by 82% and also suppressed outward currents, whereas the D127K mutation caused loss of observable single channel currents. The forgoing mutations, except for D127K, which could not be evaluated, ameliorated suppression of inwardly directed single channel currents by extracellular Ca2+. The PCa/PCs of 3.8 previously reported for the 5-HT3A(QDA) construct was reduced to 0.13 and 0.06 by the D127N and D127K mutations, respectively, with lesser, but clearly significant, effects caused by the D113N (1.04) and D113K (0.60) substitutions. Charge selectivity between monovalent cations and anions (PNa/PCl) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT3A receptor that markedly influence γ, PCa/PCs, and additionally the suppression of γ by Ca2+. The determinants of single channel conductance (γ) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that γ is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp113 and Asp127) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT3A) receptor (5-HT3A(QDA)) upon γ, modulation of the latter by extracellular Ca2+, and the permeability of Ca2+ with respect to Cs+ (PCa/PCs). Mutations neutralizing (Asp → Asn), or reversing (Asp → Lys), charge at the 113 locus decreased inward γ by 46 and 58%, respectively, but outward currents were unaffected. The D127N mutation decreased inward γ by 82% and also suppressed outward currents, whereas the D127K mutation caused loss of observable single channel currents. The forgoing mutations, except for D127K, which could not be evaluated, ameliorated suppression of inwardly directed single channel currents by extracellular Ca2+. The PCa/PCs of 3.8 previously reported for the 5-HT3A(QDA) construct was reduced to 0.13 and 0.06 by the D127N and D127K mutations, respectively, with lesser, but clearly significant, effects caused by the D113N (1.04) and D113K (0.60) substitutions. Charge selectivity between monovalent cations and anions (PNa/PCl) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT3A receptor that markedly influence γ, PCa/PCs, and additionally the suppression of γ by Ca2+. The pentameric ligand-gated ion channel (pLGIC) 4The abbreviations used are: pLGIC, pentameric ligand gated ion channel; 5-HT, 5-hydroxytryptamine; 5-HT3, 5-HT type 3; nACh, nicotinic acetylcholine; ECD, extracellular domain; TM, transmembrane; MA, membrane-associated; pS, picosiemens; GHK, Goldman-Hodgkin-Katz; ANOVA, analysis of variance; γ, single channel conductance; RI, rectification index. superfamily in eukaryotes can be subdivided into two functional groups based upon the charge selectivity of the ion channel in the open state. The excitatory 5-hydroxytryptamine type 3 (5-HT3) and nicotinic acetylcholine (nACh) receptors, together with the zinc-activated channel, are non-selective cation channels that are permeable to Na+, K+, and, to varying degrees, Ca2+ and Mg2+ (1Peters J.A. Cooper M.A. Carland J.E. Livesey M.R. Hales T.G. Lambert J.J. J. Physiol. 2010; 588: 587-596Crossref PubMed Scopus (38) Google Scholar, 2Thompson A.J. Lester H.A. Lummis S.C. Q. Rev. Biophys. 2010; 43: 449-499Crossref PubMed Scopus (260) Google Scholar, 3Sine S.M. Wang H.L. Hansen S. Taylor P. J. Mol. Neurosci. 2010; 40: 70-76Crossref PubMed Scopus (23) Google Scholar). The inhibitory γ-aminobutyric acid type A (GABAA) and glycine receptors are anion-selective and conduct mainly Cl− and HCO3− under physiological conditions (4Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (162) Google Scholar, 5Absalom N.L. Schofield P.R. Lewis T.M. Neurochem. Res. 2009; 34: 1805-1815Crossref PubMed Scopus (16) Google Scholar, 6Jensen M.L. Schousboe A. Ahring P.K. J. Neurochem. 2005; 92: 217-225Crossref PubMed Scopus (73) Google Scholar). All eukaryotic pLGIC subunits possess three topologically and functionally homologous domains (7Karlin A. Nat. Rev. Neurosci. 2002; 3: 102-114Crossref PubMed Scopus (781) Google Scholar, 8Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1082) Google Scholar, 9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar, 10Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (426) Google Scholar, 11Tsetlin V. Hucho F. Curr. Opin. Pharmacol. 2009; 9: 306-310Crossref PubMed Scopus (28) Google Scholar): (i) an extracellular domain (ECD), (ii) the transmembrane domain, and (iii) an intracellular domain. The ECD is rich in β-sheet and loop motifs and contributes to the ligand binding site, which is located at subunit interfaces, and the extracellular vestibule of the ion channel. The transmembrane domain comprises four α-helical stretches (TM1–TM4) organized such that TM1, TM3, and TM4 shield TM2 from the lipid bilayer (8Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1082) Google Scholar, 9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar). The TM regions are linked by extracellular (TM2-TM3) and intracellular (TM1-TM2 and TM3-TM4) loops. TM2 forms the lining of the transmembrane portion of the ion channel and contains the channel gate (9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar). The TM3-TM4 linker (the large intracellular loop) forms the bulk of the intracellular domain and is the least conserved region of the subunit peptide across the pLGIC receptor family. The structure of the intracellular domain is largely unresolved, apart from an α-helical region (the MA stretch) that is located immediately N-terminal to TM4 in images of the Torpedo nACh receptor obtained by cryoelectron microscopy (cryo-EM) (9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar, 12Kukhtina V. Kottwitz D. Strauss H. Heise B. Chebotareva N. Tsetlin V. Hucho F. J. Neurochem. 2006; 97: 63-67Crossref PubMed Scopus (30) Google Scholar). It is axiomatic that residues located within TM2 and the TM1-TM2 linker influence profoundly (i) single channel conductance (γ), (ii) charge selectivity, and (iii) for cation selective channels, di- versus monovalent cation selectivity (13Imoto 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, 14Galzi J.L. Devillers-Thiéry A. Hussy N. Bertrand S. Changeux J.P. Bertrand D. Nature. 1992; 359: 500-505Crossref PubMed Scopus (344) Google Scholar, 15Bertrand D. Galzi J.L. Devillers-Thiéry A. Bertrand S. Changeux J.P. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 6971-6975Crossref PubMed Scopus (342) Google Scholar) (reviewed in Ref. 4Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (162) Google Scholar). However, several recent studies have revealed that regions homologous to the MA stretch of the Torpedo nACh receptor are additional determinants of γ in 5-HT3, nACh α4β2, and α1 glycine receptors (16Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (233) Google Scholar, 17Hales T.G. Dunlop J.I. Deeb T.Z. Carland J.E. Kelley S.P. Lambert J.J. Peters J.A. J. Biol. Chem. 2006; 281: 8062-8071Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Deeb T.Z. Carland J.E. Cooper M.A. Livesey M.R. Lambert J.J. Peters J.A. Hales T.G. J. Biol. Chem. 2007; 282: 6172-6182Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 19Carland J.E. Cooper M.A. Sugiharto S. Jeong H.J. Lewis T.M. Barry P.H. Peters J.A. Lambert J.J. Moorhouse A.J. J. Biol. Chem. 2009; 284: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) (reviewed in Ref. 1Peters J.A. Cooper M.A. Carland J.E. Livesey M.R. Hales T.G. Lambert J.J. J. Physiol. 2010; 588: 587-596Crossref PubMed Scopus (38) Google Scholar). In the case of the 5-HT3A receptor, this region also contributes to di- versus monovalent cation selectivity but not charge selectivity (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The 4 Å resolution structure of the Torpedo nACh receptor obtained by cryo-EM and image reconstruction (8Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1082) Google Scholar, 9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar) reveals that the MA stretch, with a contribution from the TM1-TM2 linker, frames intracellular fenestrations through which ions must flow to enter or exit the cytoplasmic vestibule of the channel, providing a structural basis for such observations. Adding to the concept of an extended permeation pathway that influences the biophysical properties of pLGICs, the wide extracellular vestibule, surrounded by the pore lining regions of the ECD, has long been theorized to localize cations to the narrower transmembrane channel (21Dani J.A. Biophys. J. 1986; 49: 607-618Abstract Full Text PDF PubMed Scopus (132) Google Scholar). Indeed, the cryo-EM images of the Torpedo nACh receptor depict an electronegative vestibule of ∼60 Å in length and maximally 20 Å in width that has been predicted to strongly influence cation conduction (9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar). Using a homology model of the closed state of the adult muscle nACh receptor ((α1)2β1γϵ), based upon the cryo-EM images of the Torpedo nACh receptor (9Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar), Wang et al. (22Wang H.L. Cheng X. Taylor P. McCammon J.A. Sine S.M. PloS Comput. Biol. 2008; 4: e41Crossref PubMed Scopus (50) Google Scholar) conducted in silico molecular dynamics simulations of the transition of Na+ ions through the conduction pathway over a time frame of 16 ns. The simulation revealed the stabilization of cations at a number of positions along the channel axis. Notably, the extracellular vestibule was highlighted to present negative charges corresponding to the α1 subunit residues Glu83 and Asp97, where cations appeared to reside before further translocation to the transmembrane portion of the channel. Numerous additional computational approaches applied to homology models of the nACh receptor indicate that the ECD acts to concentrate monovalent cations over anions (e.g. see Refs. 23Meltzer R.H. Vila-Carriles W. Ebalunode J.O. Briggs J.M. Pedersen S.E. Biophys. J. 2006; 91: 1325-1335Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar and 24Song C. Corry B. Biochim. Biophys. Acta. 2009; 1788: 1466-1473Crossref PubMed Scopus (15) Google Scholar). By contrast, a homology model of the GABAA receptor indicates the ECD to concentrate anions in preference to cations (25O'Mara M. Cromer B. Parker M. Chung S.H. Biophys. J. 2005; 88: 3286-3299Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Direct experimental evidence for a role of the extracellular vestibule of human muscle nACh receptors in setting γ has been provided by Hansen et al. (26Hansen S.B. Wang H.L. Taylor P. Sine S.M. J. Biol. Chem. 2008; 283: 36066-36070Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), who made mutations within a negatively charged ring corresponding to Asp97 in the α1 subunit. Mutation of the residues at this locus to lysine in all subunits caused a maximal reduction of γ to ∼20% of control, as observed in recordings performed on cell-attached patches. In this study, we investigate the influence of Asp113 and Asp127 of the 5-HT3A receptor, which align with Glu83 and Asp97 of the α1 nACh receptor subunit, respectively, upon γ, single current channel rectification, ion selectivity, and modulation of γ by extracellular Ca2+. The analyses were performed in the background of a high γ mutant of the human 5-HT3A receptor (5-HT3A(QDA)), because the wild-type 5-HT3A receptor has a γ value too small to quantify by conventional single channel recording techniques (1Peters J.A. Cooper M.A. Carland J.E. Livesey M.R. Hales T.G. Lambert J.J. J. Physiol. 2010; 588: 587-596Crossref PubMed Scopus (38) Google Scholar, 16Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (233) Google Scholar, 27Brown A.M. Hope A.G. Lambert J.J. Peters J.A. J. Physiol. 1998; 507: 653-665Crossref PubMed Scopus (104) Google Scholar). The mutations introduced into the large intracellular loop of the human 5-HT3A receptor to generate the 5-HT3A(QDA) receptor construct are R432Q, R436D, and R440A (16Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (233) Google Scholar). We demonstrate that the extracellular channel vestibule within this model cationic pLGIC strongly influences γ and is also an important determinant of relative permeability to Ca2+ and the modulation of γ by extracellular Ca2+. A preliminary account of some of the results has appeared in abstract form (28.Livesey, M., Cooper, M., Lambert, J., Peters, J., (2009) Proceedings of the British Pharmacological Society, www.pa2online.org/abstracts/Vol7Issue2abst037P.pdf.Google Scholar). The methods employed to generate mutant constructs within the 5-HT3A(QDA) cDNA background and to transiently transfect tsA-201 cells with cDNA constructs were as detailed previously (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The mutations generated in this study were 5-HT3A(QDA) D113N, 5-HT3A(QDA) D113K, 5-HT3A(QDA) D127N, and 5-HT3A(QDA) D127K. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 100 μg/ml streptomycin, and 100 units/ml penicillin. Cells were incubated at 37 °C for 18–96 h in 95% air, 5% CO2 at 100% humidity prior to electrophysiological recordings. The whole-cell and outside-out patch configurations of the patch clamp technique were used to record macroscopic and single channel currents evoked by 5-HT, respectively. Patch electrodes for both modes were filled with a solution comprising 140 mm CsCl, 0.1 mm CaCl2, 1.1 mm EGTA, 10 mm HEPES, pH 7.2 (adjusted with 1 m CsOH; final [Cs+]o = 143 mm). The intracellular free calcium concentration of this solution was estimated to be 10 nm (29Fenwick E.M. Marty A. Neher E. J. Physiol. 1982; 331: 577-597Crossref PubMed Scopus (580) Google Scholar). The recording chamber comprised a 35-mm Petri dish on which the cells were cultivated. Using a gravity-feed system, the chamber was superfused routinely at ∼5 ml/min, at room temperature (20–23 °C), with extracellular solutions of the compositions described below. Single channel currents evoked by pressure-applied 5-HT (10 μm) were recorded in an extracellular solution (E1) consisting of 142.8 mm NaCl, 0.1 mm MgCl2, 0.1 mm CaCl2, 10 mm glucose, 10 mm HEPES, pH 7.2 (adjusted by 1 m NaOH; final [Na+]o = 146 mm). Whole-cell recording was used to determine the reversal potentials (E5-HT) of macroscopic currents elicited by 5-HT (10 μm) by means of a voltage-ramp protocol using WinWCP V3 9.6 electrophysiology software (J. Dempster, Department of Physiology and Pharmacology, University of Strathclyde, UK; available on the World Wide Web). Unless stated otherwise, E5-HT was determined via a protocol wherein the membrane potential was initially set at −60 mV, and a macroscopic current response to 5-HT was elicited. During the plateau of the current response, the membrane potential was stepped from −60 to −100 mV for 100 ms and subsequently ramped to +80 mV within 1 s. Care was taken to ensure that the current amplitude at the beginning and termination of the voltage ramp protocol were similar. An identical voltage ramp in the absence of 5-HT served to determine the leakage current and was subtracted from the currents evoked in the presence of 5-HT to generate the current-voltage (I-V) relationship specifically attributable to the 5-HT-evoked conductance. E5-HT was determined from such leak-subtracted currents. In order to determine the permeability of Na+ relative to Cs+ (PNa/PCs), E5-HT was determined in solution E1, and the permeability ratio was calculated from the Goldman-Hodgkin-Katz (GHK) (voltage) equation, E5-HT=RTFln(PNa/PCs)[Na+]o[Cs+]i(eq.1) where R, T, and F have their usual meaning, and [Na+]o and [Cs+]i are the calculated activities of extracellular Na+ and internal Cs+ ions. This equation ignores the very small error anticipated due to the presence of low concentrations of permeant divalent ions (e.g. Ca2+) within the extra- or intracellular solutions. To calculate the permeability of Ca2+ relative to Cs+ (PCa/PCs), E5-HT was determined in solution E2, comprising CaCl2 100 mm, glucose 10 mm, l-histidine 5 mm (pH 7.2), and the ratio was derived from a modified GHK (voltage) equation (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 30Lewis C.A. J. Physiol. 1979; 286: 417-445Crossref PubMed Scopus (277) Google Scholar), E5-HT=RTFln4(PCa'/PCs)[Ca2+]o[Cs+]i(eq.2) where [Ca2+]o is the external activity of Ca2+, [Cs+]i is the internal activity of Cs+, and P′Ca/PCs is a modified term relating the permeability of Ca2+ to Cs+. Substituting for P′Ca/PCs, the above can be rewritten as follows. expE5-HTF/RT=4PCa[Ca2+]oPCs(1+expE5-HTF/RT)[Cs+]i(eq.3) For simplicity, we routinely refer to ion concentrations rather than activities throughout, although the latter were employed in the calculations of relative permeabilities. To examine modulation of γ by extracellular Ca2+, single channel recordings and determinations of E5-HT were also made in extracellular solutions (E3, E4, and E5) containing variable [CaCl2]o (0.1, 1, and 10 mm, respectively) but constant [NaCl]o (95 mm), l-histidine (5 mm), and glucose 10 (mm), pH 7.2. The osmolarity of such solutions was held constant by the addition of appropriate amounts of sucrose. An extracellular solution (E6) containing 20 mm NaCl, 0.1 mm MgCl2, 0.1 mm CaCl2, 10 mm glucose, 239.4 mm sucrose, 5 mm l-histidine, pH 7.2 (adjusted by 1 m HCl), in addition to solutions E1 and E3, was used to perform dilution potential measurements in order to determine relative permeability to chloride. Liquid junction potentials between the pipette tip and extracellular solutions were measured according to Fenwick et al. (29Fenwick E.M. Marty A. Neher E. J. Physiol. 1982; 331: 577-597Crossref PubMed Scopus (580) Google Scholar) and were corrected post hoc. In brief, the zero current voltage was initially determined with the intracellular solution present within both the patch pipette and the bath. The extracellular solutions (E1–E6) used in the experiments then replaced the intracellular solution, and the change in zero current voltage (i.e. the liquid junction potential) was noted. A salt bridge consisting of 3 m KCl in 4% (w/v) agar connected the bath to the reference electrode, eliminating changes in the potential of the latter during this procedure. Single channel currents were low pass-filtered offline at 1 kHz, digitized at 10 kHz via a DigiData 1302A (Axon Instruments) interface. Using the WinEDR V2 7.6 Electrophysiology Data Recorder (J. Dempster, Department of Physiology and Pharmacology, University of Strathclyde, UK; available on the World Wide Web), single channel current amplitude histograms were constructed from sections of single channel activity in which unitary events predominated, as described in detail previously (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The γ value is routinely reported as the chord conductance (i.e. γ = i/(Vm − E5-HT), where i is single channel current amplitude, Vm is the holding potential (including liquid junction potential correction), and E5-HT is the reversal potential of the agonist-evoked macroscopic response determined as described above under the appropriate ionic conditions. In the case of the 5-HT3A(QDA) D127K mutant, single channel currents could not be resolved directly in recording from outside-out membrane patches. Thus, we estimated γ by fluctuation analysis of whole-cell current evoked by 5-HT (1 μm) as previously described by us (16Kelley S.P. Dunlop J.I. Kirkness E.F. Lambert J.J. Peters J.A. Nature. 2003; 424: 321-324Crossref PubMed Scopus (233) Google Scholar). A rectification index (RI) is used to describe the degree of single channel current rectification. The RI was calculated as follows, RI=slope γ(positive V)slope γ(negative V)(eq.4) where slope γ(positive V) and slope γ(negative V) were obtained from obtaining the slope of the line of best fit plotted to data points from positive and negative holding potentials, respectively. An RI value of 1 indicates a linear/ohmic i-V relationship. An RI of >1 indicates outward rectification, and RI <1 indicates inward rectification. Data are presented as mean ± S.E. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with the post hoc Dunnett's or Dunn's test as appropriate. p < 0.05 was considered to be significant. Asp113 is predicted from a homology model of the human 5-HT3A receptor (18Deeb T.Z. Carland J.E. Cooper M.A. Livesey M.R. Lambert J.J. Peters J.A. Hales T.G. J. Biol. Chem. 2007; 282: 6172-6182Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) to reside approximately one-third of the distance from the entrance to the extracellular vestibule to the extracellular border of TM2. Asp113 is homologous to the Torpedo nACh receptor α1 subunit Glu83 locus and is modeled as forming a ring of negative charge within the apical extracellular vestibule (Fig. 1). Asp113 was mutated to either a neutral asparagine or a positively charged lysine, within the 5-HT3A(QDA) construct, yielding 5-HT3A(QDA) D113N or 5-HT3A(QDA) D113K, respectively. At a holding potential of −80 mV in extracellular (E1) and intracellular solutions containing Na+ and Cs+ as the principal charge carriers respectively, 5-HT (10 μm) applied by pressure to outside-out patches excised from tsA-201 cells expressing 5-HT3A(QDA) D113N mutant receptors elicited clearly resolvable single channel events. By comparison with the 5-HT3A(QDA) receptor construct previously characterized under identical ionic conditions (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), inwardly directed single channel currents mediated by the 5-HT3A(QDA) D113N construct are effectively halved in amplitude, the chord conductance (γ) being reduced from 41.2 pS to 22.2 ± 0.3 pS (Fig. 2, A and B, and Table 1). The 5-HT3A(QDA) D113K receptor displayed a γ value that was further reduced to 17.1 ± 0.2 pS (Fig. 2, A and B, and Table 1). Although the γ value of the D113N and D113K 5-HT3A(QDA) constructs did not differ strikingly, the reduction caused by reversal, rather than neutralization, of charge was significantly greater (Table 1).FIGURE 2Neutralization, or reversal, of charge at the Asp113 locus depresses inwardly directed single channel currents. Single channel events, elicited by 5-HT (10 μm), were recorded from outside-out patches excised from tsA-201 cells transfected with the construct of interest. The Cs+-based pipette solution and extracellular solution E1 were utilized. A, exemplar single channel events recorded at −80 and +80 mV from the 5-HT3A(QDA), 5-HT3A(QDA) D113N, and 5-HT3A(QDA) D113K mutant receptors. B, i-V profiles for the 5-HT3A(QDA) (solid circles), 5-HT3A(QDA) D113N (open circles), and 5-HT3A(QDA) D113K (inverted triangles) receptors. Note that mutation of the Asp113 residue does not impact upon outwardly directed single channel currents. Data points are the mean of a minimum of three observations made from separate patches, and error bars, where visible, indicate S.E. Data for the 5-HT3A(QDA) receptor are from Livesey et al. (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1The influence of mutations within the extracellular domain (ECD) upon γ and single channel current rectificationReceptor constructsγnRectification indexpS5-HT3A(QDA)41.2 ± 1.2aData reported previously by Livesey et al. (20).81.125-HT3A(QDA) D113N22.2 ± 0.3bSignificantly different (p < 0.001) from the 5-HT3A(QDA) mutant receptor, as determined by one-way ANOVA with post hoc Dunnett's test.91.75-HT3A(QDA) D113K17.1 ± 0.2bSignificantly different (p < 0.001) from the 5-HT3A(QDA) mutant receptor, as determined by one-way ANOVA with post hoc Dunnett's test.,cSignificantly different (p < 0.001) from the 5-HT3A(QDA) D113N mutant receptor construct, as determined by unpaired t test.82.635-HT3A(QDA) D127N7.4 ± 0.1bSignificantly different (p < 0.001) from the 5-HT3A(QDA) mutant receptor, as determined by one-way ANOVA with post hoc Dunnett's test.33.185-HT3A(QDA) D127K1.6 ± 0.3bSignificantly different (p < 0.001) from the 5-HT3A(QDA) mutant receptor, as determined by one-way ANOVA with post hoc Dunnett's test.6NDa Data reported previously by Livesey et al. (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).b Significantly different (p < 0.001) from the 5-HT3A(QDA) mutant receptor, as determined by one-way ANOVA with post hoc Dunnett's test.c Significantly different (p < 0.001) from the 5-HT3A(QDA) D113N mutant receptor construct, as determined by unpaired t test. Open table in a new tab Single channel current-voltage (i-V) relationships were generated by recording unitary currents evoked by 5-HT (10 μm) from outside-out patches at holding potentials between −100 and +100 mV in increments of 20 mV. For the 5-HT3A(QDA) receptor construct, the single channel current i-V relationship is linear at negative potentials with a slope γ of 41 pS (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, at positive potentials, γ increases to 46 pS, indicative of modest outward rectification quantified as an RI value (see “Experimental Procedures”) of 1.12 (20Livesey M.R. Cooper M.A. Deeb T.Z. Carland J.E. Kozuska J. Hales T.G. Lambert J.J. Peters J.A. J. Biol. Chem. 2008; 283: 19301-19313Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (Fig. 2B). Under identical ionic conditions, the i-V relationships obtained for the 5-HT3A(QDA) D113N and the 5-HT3A(QDA) D113K receptor constructs reveal that both mutations enhance outward rectification, the RI values being 1.7 and 2.63, respectively (Table 1). Notably, either mutation reduced only inwardly (and not outwardly) directed single channel currents (Fig. 2B). Such results are consistent with a scheme whereby the ring of negativity formed by the Asp113 residues acts via simple coulombic attraction to raise the availability of cations within the receptor extracellular vestibule for translocation to deeper regions of the pore. Neutralization or reversal of charge at this locus would be anticipated to reduce the local concentration of cations within the extracellular vestibule, leading to the emergence of outward rectification. Notably, the effect of neutralizing or reversing the charge at the Asp113 position was increased when [Na+]o was reduced to 95 mm (see below) (see Fig. 5). In comparison with the 4" @default.
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• W2035499881 title "Rings of Charge within the Extracellular Vestibule Influence Ion Permeation of the 5-HT3A Receptor" @default.
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