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.     

The extracellular linker of muscle acetylcholine receptor channels is a gating control element

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.     

Molecular basis for zinc potentiation at strychnine-sensitive glycine receptors

The divalent cation Zn(2+) is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn(2+). Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn(2+) potentiation site on GlyRs and account for the differential sensitivity of GlyR alpha(1) and GlyR alpha(2) to Zn(2+) potentiation. In addition, juxtaposed to this Zn(2+) site, which is located externally on the N-terminal domain of the alpha subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn(2+) potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn(2+) binds to a site on the extracellular outer face of the GlyR alpha subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.     

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.     

Role of the conserved lysine residue in the middle of the predicted extracellular loop between M2 and M3 in the GABA(A) receptor.

In alpha1, beta2, and gamma2 subunits of the gamma-aminobutyric acid A (GABA(A)) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in alpha1 and beta2 subunits resulted each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the gamma subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the alpha1 subunit or the beta2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the alpha and beta subunits. Binding of [3H]Ro 15-1788 was unaffected by the mutation in the alpha subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the alpha1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the alpha1 and beta2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

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.     

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an alpha(2)betaepsilondelta composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15' of the M1 domain is phenylalanine in alpha subunits while it is isoleucine in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant alpha subunit (alphaF15'I) as well as those containing the reverse mutations in the non-alpha subunits (betaI15'F, deltaI15'F, and epsilonI15'F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the beta subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of betaI15'F of AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant in the diliganded receptor, and a significant increase opening in the absence of agonist. Mutations at betaI15' showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an approximately 70% closed-state-like environment for the beta15' position at the transition state of gating. The overall results identify position 15' as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.     

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.     

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.     

Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers.

We have expressed glycine receptor (GlyR) alpha and beta subunit cDNAs in HEK-293 cells to study the functional properties of homo- versus hetero-oligomeric GlyR channels. Dose-response curves of whole-cell currents in cells expressing alpha 1 subunits revealed an average Hill coefficient of h = 4.2. Co-expression with the beta subunit markedly increased glycine-gated whole-cell currents, which now exhibited a mean Hill coefficient of only h = 2.5. For alpha 1, alpha 2 and alpha 3 homo-oligomers, the main-state single-channel conductances were 86, 111 and 105 pS, respectively, recorded at symmetrical Cl- concentrations of 145 mM. The mutant alpha 1 G221A gave rise to a main-state of 107 pS. This indicates that the main-state of alpha homo-oligomers depends on residue 221 which is located within transmembrane segment M2. Importantly, the main-state conductances of alpha 1/beta, alpha 2/beta and alpha 3/beta hetero-oligomers were only 44, 54 and 48 pS, respectively. The latter values are similar to those found in spinal neurons, suggesting that native GlyRs are predominantly alpha/beta hetero-oligomers. Co-expression of alpha 1 with mutant beta subunits revealed that residues within and close to segment M2 of the beta subunit determine the conductance differences between homo- and hetero-oligomers.     

Functional asymmetry of the conserved cystine loops in alphabetagamma GABA A receptors revealed by the response to GABA activation and drug potentiation

Ligand-gated ion channels respond to specific neurotransmitters by transiently opening an integral membrane ion-selective pore, allowing ions to move down their electrochemical gradient. A distinguishing feature of all members of the ligand-gated ion channel superfamily is the presence of a 13-amino acid disulfide loop (Cys-loop) in the extracellular ligand-binding domain. Structural data derived from the acetylcholine receptor place this loop at the interface between the ligand-binding domain and the transmembrane pore-forming domain where it is ideally located to participate in coupling ligand binding to channel opening. We have introduced specific mutations into a conserved motif at the mid-point of the Cys-loop of the GABA A receptor subunits alpha1, beta2 and gamma2S where the sequence reads aromatic, proline, aliphatic (ArProAl motif). Receptors carrying a mutation in the Cys-loop of one of their subunits were expressed in L929 cells and responses to both GABA and drugs were assessed using the whole-cell patch clamp technique. Drug potentiation and direct activation were significantly enhanced by mutations in this Cys-loop but these effects were subunit-dependent. Currents in response to agonists were larger when mutations were carried in the alpha and beta subunits but not in the gamma subunit. In contrast, potentiation of current responses by diazepam, etomidate and pentobarbital were all enhanced when mutations were carried in the alpha and gamma subunits, but not the beta subunit. Since the disruption of interactions mediated through the ArProAl motif enhances the mutant receptor's response to both agonist and drugs we suggest that this motif in the Cys-loop of the wild-type receptor participates in interactions that create activation barriers to conformational changes during channel gating.     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor (GlyR) alpha1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His107 from one subunit and His109 from an adjacent subunit. This was tested by co-expressing alpha1 subunits containing the H107A mutation with alpha1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha1 subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha1beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha1 homomers. The binding of zinc at the interface between adjacent alpha1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.     

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alphabeta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alphabeta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alphabeta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alphabeta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting 'competitive' antagonist) binds to this residue.     

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.     

The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.     

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.     

Differential protein mobility of the gamma-aminobutyric acid, type A, receptor alpha and beta subunit channel-lining segments

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.     

The zona pellucida-initiated acrosome reaction: defect due to mutations in the sperm glycine receptor/Cl(-) channel

The mammalian sperm acrosome reaction (AR) is essential to fertilization, and the egg zona pellucida (ZP) is generally believed to be an in vivo initiator of the fertilizing sperm AR. Previously a neuronal glycine receptor/Cl(-) channel (GlyR) was detected on the plasma membrane of mammalian sperm and earlier pharmacological studies suggested that this receptor/channel is important to the ZP-initiated AR. Here, sperm from mice with mutations in the neuronal GlyR alpha or beta subunits (spasmodic and spastic) were shown to be deficient in their ability to undergo the AR initiated in vitro by glycine or by solubilized ZP from mouse eggs. However, both spontaneous and calcium ionophore (A23187)-initiated AR were unaffected. The ZP-initiated AR in wild-type sperm was maximal after 2 h of capacitation, but capacitation of sperm from spasmodic mice for up to 3 h did not result in significant ZP-initiated AR. Similar results were observed when sperm from wild-type and spastic mice were compared. Testis from mice with the beta subunit mutation contained truncated beta subunit mRNAs. Moreover, a monoclonal antibody against GlyR completely blocked ZP initiation of AR in normal mouse sperm. Our results are consistent with an essential role for the sperm GlyR in the ZP-initiated AR.     

Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function

Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function.