Closed-state cross-linking of adjacent beta1 subunits in alpha1beta1 GABAa receptors via introduced 6' cysteines

The pore structural changes associated with Cys-loop receptor gating are currently the subject of considerable interest. Several functional approaches have shown that surface exposure of pore-lining side chains does not change significantly during activation. However, a disulfide trapping study on alpha1(T6'C)beta1(T6'C) gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs), which showed that adjacent beta subunits cross-link in the open state only, concluded that channel gating is mediated by beta subunits contra-rotating through a summed angle of approximately 120 degrees. Such a large rotation is not easily reconciled with other evidence. The present study initially sought to investigate an observation that appeared inconsistent with the rotation model: namely that alpha1(T6'C)beta1(T6'C) GABA(A)Rs expressed in HEK293 cells form 6' cysteine-mediated disulfide bonds in the closed state. On the basis of electrophysiological and Western blotting experiments, we conclude that adjacent beta(T6'C) subunits dimerise efficiently in the closed state via cross-links between their respective 6' cysteines and that this locks the channels closed. This questions the beta subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that beta subunit 6' cysteines move into sufficiently close proximity for disulfide formation via relatively large amplitude random thermal motions that appear to be a unique feature of beta subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous beta subunit movements or asymmetries in the movements of adjacent beta subunits during activation are essential for pore opening. Our results identify a novel feature of GABA(A)R gating that may be important for understanding its activation mechanism.     

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha1, alpha2, alpha1beta and alpha2beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha2beta GlyR relative to the alpha2 GlyR but not in the alpha1beta GlyR relative to the alpha1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha2beta GlyR was transferred to the alpha1beta GlyR by the G2'A (alpha1 to alpha2 subunit) substitution. In addition, the alpha1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.     

Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.     

Structure-activity analysis of ginkgolide binding in the glycine receptor pore

Ginkgolides, active constituents of Ginkgo biloba extracts, potently block the glycine receptor chloride channel (GlyR). Ginkgolides A, B, C and J are structurally similar, varying only by the presence or absence of oxygens at their R1 and R2 positions. The aim of this study was to understand how variable ginkgolide groups bind to pore-lining 2' and 6' residues in the α1 GlyR. Ginkgolide potency was not affected by G2'A or G2'S mutations, suggesting 2' residues are not important for ginkgolide coordination. Analysis of the α1^T6'S GlyR suggests that ginkgolides bind to this receptor via hydrogen bonds between T6'S and ginkgolide R1 hydroxyls. The abolition of block by the T6'A and T6'V mutations but not by the T6'S mutation implies the existence a second transmembrane domain α-helical kink formed by hydrogen bonding between 6' threonine and serine sidechains and backbone carbonyl oxygens. We also found that ginkgolide A binds in different orientations in the closed and open states of a mutant GlyR, possibly reflecting its enhanced flexibility relative to other ginkgolides. Together these results indicate that small variations in ginkgolide structure or pore structure can lead to drastic potency variations. This property may be exploited to create improved pharmacological probes for discriminating among anionic Cys-loop receptor isoforms with 6' structural variations.     

NMR structures of the second transmembrane domain of the human glycine receptor alpha(1) subunit: model of pore architecture and channel gating

Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR alpha(1) subunit are unique in that they form functional homopentameric channels with conductance characteristics nearly identical to those of an authentic receptor (Opella, S. J., J. Gesell, A. R. Valente, F. M. Marassi, M. Oblatt-Montal, W. Sun, A. F. Montiel, and M. Montal. 1997. Chemtracts Biochem. Mol. Biol. 10:153-174). Using NMR and circular dichroism (CD), we determined the high-resolution structures of the TM2 segment of human alpha(1) GlyR and an anesthetic-insensitive mutant (S267Y) in dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. The NMR structures showed right-handed alpha-helices without kinks. A well-defined hydrophilic path, composed of side chains of G2', T6', T10', Q14', and S18', runs along the helical surfaces at an angle approximately 10-20 degrees relative to the long axis of the helices. The side-chain arrangement of the NMR-derived structures and the energy minimization of a homopentameric TM2 channel in a fully hydrated DMPC membrane using large-scale computation suggest a model of pore architecture in which simultaneous tilting movements of entire TM2 helices by a mere 10 degrees may be sufficient to account for the channel gating. The model also suggests that additional residues accessible from within the pore include L3', T7', T13', and G17'. A similar pore architecture and gating mechanism may apply to other channels in the same superfamily, including GABA(A), nACh, and 5-HT(3) receptors.     

Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships

GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundstr?m, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to alpha(1) GlyR and rho(1) GABAR, the rounded end of PTX accepts several H-bonds from Thr(6')s, while the elongated end enters ring R(2'). The lack of H-bond donors on the side chains of Phe(6')s (beta GlyR) and Met(6')s (rho(2) GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro(2')s/Ala(2')s in GABARs, but fit a more hydrophilic ring with Gly(2')s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations.     

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.     

A proposed structural basis for picrotoxinin and picrotin binding in the glycine receptor pore

Picrotoxin, an antagonist of structurally-rated GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs), is an equimolar mixture of picrotoxinin (PTXININ) and picrotin (PTN). These compounds share a common structure except that PTN contains a slightly larger dimethylmethanol in place of the PTXININ isopropenyl group. Although the homomeric alpha1 GlyR is equally sensitive to both compounds, we show here that homomeric alpha2 and alpha3 GlyRs, like most GABA(A)Rs, are selectively inhibited by PTXININ. As conservative mutations to pore-lining 6' threonines equally affect the sensitivity of the alpha1 GlyR to both compounds, we conclude that PTXININ and PTN bind to 6' threonines by hydrogen bonding with exocyclic oxygens common to both molecules. In contrast, substitution of the 2' pore-lining glycine by serine selectively reduces PTN sensitivity, whereas the introduction of 2' alanines selectively increases PTXININ sensitivity. These results define the orientation of PTXININ and PTN binding in the alpha1 GlyR pore and allow us to conclude that the relatively reduced sensitivity of PTN at GABA(A)Rs and alpha2 and alpha3 GlyRs is due predominantly to its larger size and reduced ability to form hydrophobic interactions with 2' alanines.     

Mechanisms for picrotoxin block of alpha2 homomeric glycine receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.     

Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action

The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.     

Different residues in the GABA(A) receptor alpha 1T60-alpha 1K70 region mediate GABA and SR-95531 actions

Although gamma-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from alpha(1)T60 to alpha(1)K70 was mutated to cysteine and expressed with wild-type beta(2) subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with alpha(1)T60C, alpha(1)D62C, alpha(1)F64C, alpha(1)R66C, alpha(1)S68C, and alpha(1)K70C. gamma-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of alpha(1)F64C, alpha(1)R66C, and alpha(1)S68C, whereas SR-95531 slowed modification of alpha(1)D62C, alpha(1)F64C, and alpha(1)R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for alpha(1)F64C and alpha(1)R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized alpha(1)F64C and alpha(1)R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of alpha(1)D62C and alpha(1)S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.     

Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel

The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.     

Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.     

Theoretical studies of the M2 transmembrane segment of the glycine receptor: models of the open pore structure and current-voltage characteristics

The pentameric glycine receptor (GlyR), a member of the nicotinicoid superfamily of ligand-gated ion channels, is an inhibitory Cl(-) channel that is gated by glycine. Using recently published NMR data of the second transmembrane segment (M2) of the human alpha1 GlyR, structural models of pentameric assemblies embedded in a lipid bilayer were constructed using a combination of experimentally determined constraints coupled with all-atom energy minimization. Based on this structure of the pentameric M2 "pore", Brownian dynamics simulations of ion permeation through this putative conducting open state of the channel were carried out. Simulated I-V curves were in good agreement with published experimental current-voltage curves and the anion/cation permeability ratio, suggesting that our open-state model may be representative of the conducting channel of the full-length receptor. These studies also predicted regions of chloride occupancy and suggested residues critical to anion permeation. Calculations of the conductance of the cation-selective mutant A251E channel are also consistent with experimental data. In addition, both rotation and untilting of the pore helices of our model were found to be broadly consistent with closing of the channel, albeit at distinct regions that may reflect alternate gates of the receptor.     

Conformational variability of the glycine receptor M2 domain in response to activation by different agonists

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective alpha1 homomeric glycine receptor (alpha1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1'E GlyR) was sufficient to convert the channels to select cations over anions with P(Cl)/P(Na) = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2'Delta mutant GlyR retained its anion selectivity (P(Cl)/P(Na) = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (P(Cl)/P(Na) = 27.9). When the A-1'E and the P-2'Delta mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (P(Cl)/P(Na) = 0.13). The SDM GlyR was also Ca(2+) permeable (P(Ca)/P(Na) = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca(2+)-permeable channel (P(Ca)/P(Na) = 0.73), without drastically altering monovalent charge selectivity (P(Cl)/P(Na) = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca(2+) permeability (P(Ca)/P(Na) = 0.92), with little effect on monovalent selectivity (P(Cl)/P(Na) = 0.19). Estimates of the minimum pore diameter of the A-1'E, SDM, SDM+R19'A, and SDM+R19'E GlyRs revealed that these pores are larger than the alpha1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.     

Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels

Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2' Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.     

Expression of the human glycine receptor alpha 1 subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel, which mediates post-synaptic inhibition in spinal cord and other brain regions. Heterologous expression of the ligand binding alpha subunits of the GlyR generates functional agonist-gated chloride channels that mimic most of the pharmacological properties of the receptor in vivo. Here, nuclear injection into Xenopus oocytes of a human alpha 1 subunit cDNA, engineered for efficient expression, was used to create GlyR channels over a wide density range, resulting in whole-cell glycine currents of 10 nA to 25 microA. Notably, the pharmacology of these channels changed at high expression levels, with the appearance of a novel receptor subpopulation of 5- to 6-fold higher apparent agonist affinity at current values > 4 microA. The low-affinity receptors were readily blocked by nM concentrations of the competitive antagonist strychnine, whereas the high-affinity receptors were more resistant to antagonism by this alkaloid. Picrotoxinin, a chloride channel blocker, inhibited both GlyR populations with equal potency. Our data suggest that receptor interactions, occurring at high receptor density, modify the agonist response of the GlyR. This phenomenon may contribute to neurotransmitter efficacy at fast synapses.     

Loose protein packing around the extracellular half of the GABA(A) receptor beta1 subunit M2 channel-lining segment

GABA(A) receptors are ligand-gated ion channels formed by the pseudosymmetrical assembly of five homologous subunits around the central channel axis. The five M2 membrane-spanning segments largely line the channel. In the present work we probed the water surface accessibility of the beta(1) subunit M2 segment using the substituted cysteine accessibility method. We assayed the reaction of the negatively charged sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS(-)), by its effect on subsequent currents elicited by EC(50) and saturating GABA concentrations. pCMBS(-), applied with GABA, reacted with 14 of the 19 residues tested. At the M2 cytoplasmic end from 2' to 6' only beta(1)A252C (2') and beta(1)T256C (6') were pCMBS(-)-reactive in the presence of GABA. We infer that the M2 segments are tightly packed in this region. Toward the extracellular half of M2 all residues from beta(1)T262C (12') through beta(1)E270C (20') reacted with pCMBS(-) applied with GABA. We infer that this region is highly mobile and loosely packed against the rest of the protein. Based on differences in pCMBS(-) reaction rates two domains can be distinguished on the putative channel-lining side of M2. A faster reacting domain includes the 2', 9', 12', 13', and 16' residues. The slower reacting face contains the 6', 10', and 14' residues. We hypothesize that these may represent the channel-lining faces in the closed and open states and that gating involves an 80-100 degrees rotation of the M2 segments. These results are consistent with the loose packing of the M2 segments inferred from the structure of the homologous Torpedo nicotinic acetylcholine receptor.