Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.     

β Subunit M2-M3 loop conformational changes are uncoupled from α1 β glycine receptor channel gating: implications for human hereditary hyperekplexia

Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2-M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2-M3 loop of the β than into the α1 subunit. This suggests that the M2-M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2-M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2-M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit.     

Structural Insights Into NMDA Ionotropic Glutamate Receptors via Molecular Modelling

Structural models have been produced for the agonist binding and transmembrane domains of two NMDA ionotropic glutamate receptors: homomeric NMDA-R2C and heteromeric NMDA-R1/R2C. These models--produced using homology modelling techniques in conjunction with distance restraints derived from the accessibility of substituted cysteines--have aided our understanding of (1) ligand selectivity and (2) channel activity. The model of the agonist binding domain of NMDA-R2C indicates that T691 forms an essential hydrogen bond with glutamate ligand. This interaction is absent in the NMDA-R1 model--where a valine replaces the threonine--explaining why NMDA-R1 binds glycine rather than glutamate. For the transmembrane region, the models suggest that a number of positive residues, located in the cytoplasmic loop between the M1 and M2 segments, create a large electrostatic energy barrier that could explain why homomeric NMDA-R2C channels are non-functional. Introducing NMDA-R1 to form heteromeric NMDA-R1/R2C channels is predicted to rescue channel activity because the corresponding region in NMDA-R1 contains negative residues that more than compensate for the electrostatic energy barrier in NMDA-R2C. These studies suggest that replacing the positively charged region in the M1-M2 loop of NMDA-R2C with the corresponding negatively charged region of NMDA-R1 could transform NMDA-R2C into a functional homomeric channel.     

The location of a closed channel gate in the GABAA receptor channel

Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABAA receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-A resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9' to 14' but the M1-M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABAA receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of alpha1Val257 (2') near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location.     

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel.

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.     

Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [³H]strychnine binding assays. In the wild-type (WT) GlyR, 1 µ M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.     

Asymmetric contribution of alpha and beta subunits to the activation of alphabeta heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.     

Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.     

GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating

The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.     

Distinct properties of glycine receptor β+/α- interface: unambiguously characterizing heteromeric interface reconstituted in homomeric protein

The glycine receptor (GlyR) exists either in homomeric α or heteromeric αβ forms. Its agonists bind at extracellular subunit interfaces. Unlike subunit interfaces from the homomeric α GlyR, subunit interfaces from the heteromeric αβ GlyR have not been characterized unambiguously because of the existence of multiple types of interface within single receptors. Here, we report that, by reconstituting β+/α- interfaces in a homomeric GlyR (αChb+a- GlyR), we were able to functionally characterize the αβ GlyR β+/α- interfaces. We found that the β+/α- interface had a higher agonist sensitivity than that of the α+/α- interface. This high sensitivity was contributed primarily by loop A. We also found that the β+/α- interface differentially modulates the agonist properties of glycine and taurine. Using voltage clamp fluorometry, we found that the conformational changes induced by glycine binding to the β+/α- interface were different from those induced by glycine binding to the α+/α- interface in the α GlyR. Moreover, the distinct conformational changes found at the β+/α- interface in the αChb+a- GlyR were also found in the heteromeric αβ GlyR, which suggests that the αChb+a- GlyR reconstitutes structural components and recapitulates functional properties, of the β+/α- interface in the heteromeric αβ GlyR. Our investigation not only provides structural and functional information about the GlyR β+/α- interface, which could direct GlyR β+/α- interface-specific drug design, but also provides a general methodology for unambiguously characterizing properties of specific protein interfaces from heteromeric proteins.     

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.     

Mechanisms for picrotoxinin and picrotin blocks of alpha2 homomeric glycine receptors

Contrary to its effect on the gamma-aminobutyric acid type A and C receptors, picrotoxin antagonism of the alpha1 homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and nonselective between the picrotoxin components picrotoxinin and picrotin. Picrotoxin antagonism of the embryonic alpha2 homomeric GlyR is known to be use-dependent and reflects a channel-blocking mechanism, but the selectivity of picrotoxin antagonism of the embryonic alpha2 homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin- and picrotoxinin-induced inhibition of currents, which were evoked by the activation of alpha2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents, picrotin had a 30 times higher IC50 than picrotoxinin. Picrotin-evoked inhibition displayed voltage dependence, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentration-dependent manner, indicating that these picrotoxin components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were co-applied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but both could be trapped at their binding site when the channel closed during glycine dissociation. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking alpha2 homomeric GlyR.     

Activation and desensitization induce distinct conformational changes at the extracellular-transmembrane domain interface of the glycine receptor

Most ligand-gated channels exhibit desensitization, which is the progressive fading of ionic current in the prolonged presence of agonist. This process involves conformational changes that close the channel despite continued agonist binding. Despite the physiological and pathological importance of desensitization, little is known about the conformational changes that underlie this process in any Cys-loop ion channel receptor. Here we employed voltage clamp fluorometry to identify conformational changes that occur with a similar time course as the current desensitization rate in both slow- and fast-desensitizing α1 glycine receptor chloride channels. Voltage clamp fluorometry provides a direct indication of conformational changes that occur in the immediate vicinity of residues labeled with environmentally sensitive fluorophores. We compared the rates of current desensitization and fluorescence changes at nine labeled extracellular sites in both wild type slow-desensitizing and mutated (A248L) fast-desensitizing glycine receptors. As labels attached to three sites at the interface between the ligand binding domain and transmembrane domain reported fluorescence responses that changed in parallel with the current desensitization rate, we concluded that they experienced local conformational changes associated with desensitization. These labeled sites included A52C in loop 2, Q219C in the pre-M1 domain, and M227C in the M1 domain. Activation and desensitization were accompanied by physically distinct conformational changes at each labeled site. Because activation is mediated by a specific reorganization of molecular interactions at the extracellular-transmembrane domain interface, we propose that desensitization is mediated by a distinct set of conformational changes that prevents this reorganization from occurring, thereby favoring channel closure.     

Evidence for distinct sodium-, dopamine-, and cocaine-dependent conformational changes in transmembrane segments 7 and 8 of the dopamine transporter

Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.     

A gating mutation in the internal pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.     

Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the α1 glycine receptor increases during glycine activation but not during ivermectin activation. This prompted the hypothesis that this signal reports a glycine-mediated conformational change not essential for activation. We tested this by investigating whether the fluorescence signal depended on whether the fluorophore was attached to a glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (−) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation.     

Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein

Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET(+)) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET(+) and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET(+)-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET(+) produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

NH(2)-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

A synthetic, channel-forming peptide, derived from the alpha-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH(2) terminus (N-K(4)-M2GlyR). In Ussing chamber experiments, apical N-K(4)-M2GlyR (250 microM) increased transepithelial short-circuit current (I(sc)) by 7.7 +/- 1.7 and 10.6 +/- 0.9 microA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K(4)-M2GlyR caused a concentration-dependent increase in I(sc) (k([1/2]) = 190 microM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K(4)-M2GlyR-mediated increases in I(sc) were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl(-) conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K(4)-M2GlyR-mediated I(sc). Whole cell membrane patch voltage-clamp studies revealed an N-K(4)-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K(4)-M2GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl(-) of 290 meq. These results indicate that N-K(4)-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.     

Mutations within the agonist-binding site convert the homomeric α1 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 µM) 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 α-subunits. Here, we examined the ZZn²⁺ sensitivity of different mutations within the agonist binding site of the homomeric α1-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that 6 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 (α1R65A, α1R131A), or of the aromatic residue phenylalanine 207 by histidine (α1F207H), converted the α1 GlyR into a chloride channel that was activated by Zn²⁺ alone. Dose-response analysis of the α1F207H GlyR disclosed an EC50 value of 1.2 µM for Zn²⁺ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the α1 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.