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 conserved Cys-loop receptor aspartate residue in the M3-M4 cytoplasmic loop is required for GABAA receptor assembly

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary gamma-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.     

The M3/M4 cytoplasmic loop of the alpha1 subunit restricts GABAARs lateral mobility: a study using fluorescence recovery after photobleaching

A crucial problem in neurobiology is how neurons are able to maintain neurotransmitter receptors at specific membrane domains. The large structural heterogeneity of gamma aminobutyric acid receptors (GABAARs) led to the hypothesis that there could be a link between GABAAR gene diversity and the targeting properties of the receptor complex. Previous studies using Fluorescence Recovery After Photobleaching (FRAP) have shown a restricted mobility in GABAARs containing the alpha1 subunit. The M3/M4 cytoplasmic loop is the region of the alpha1 subunit with the lowest sequence homology to other subunits. Therefore, we asked whether the M3/M4 loop is involved in cytoskeletal anchoring and GABAAR clustering. A series of alpha1 chimeric subunits was constructed: alpha1CH (control subunit), alpha1CD (Cytoplasmic loop deleted), alpha1CD2, and alpha1CD3 (alpha1 with the M3/M4 loop from the alpha2 and alpha3 subunits, respectively). Our results using FRAP indicate an involvement of the M3/M4 cytoplasmic loop of the alpha1 subunit in controlling receptor lateral mobility. On the other hand, inmunocytochemical approaches showed that this domain is not involved in subunit targeting to the cell surface, subunit-subunit assembly, or receptor aggregation.     

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.     

Improved gating of a chimeric alpha7-5HT3A receptor upon mutations at the M2-M3 extracellular loop

Acetylcholine-evoked currents of the receptor chimera alpha7-5HT3A V201 expressed in Xenopus oocytes are strikingly small when compared to the amount of alpha-bungarotoxin binding sites detected at the oocyte membrane. Since the chimeric receptor is made of the extracellular N-terminal region of the rat alpha7 nicotinic acetylcholine receptor and the C-terminal region of the mouse 5-HT3A receptor, which includes the ion channel, we hypothesized that communication between these two regions was not optimal. Here, we show that mutating to aspartate several adjacent positions in the M2-M3 extracellular linker increases current amplitudes to different extents, thus confirming the important role of this region on receptor gating.     

Characterization of newly cloned variant of rat glycine receptor alpha1 subunit

Responses to glycine, a major inhibitory neurotransmitter within the nervous system, are mediated by glycine receptors (GlyRs). Here, we report the cloning and analysis of a novel splicing variant of the GlyRalpha1 subunit. This variant, named GlyRalpha1del, has a truncated cytoplasmic region between transmembrane domains (TM)3 and TM4, and compared to other variants, the truncation is contributed by a different acceptor site in exon 9. We transfected GlyRalpha1 or GlyRalpha1del into HEK293 cells, and then examined the glycine-activated currents using a whole-cell patch-clamp recording technique. Maximal currents and current-voltage relationships showed no clear difference between GlyRalpha1del and GlyRalpha1. Moreover, dose-response curves indicated that the EC50 values for glycine differed significantly between the two GlyRalpha1 derivatives, although their Hill coefficients were similar. When present with other isoforms, GlyRalpha1del might alter the response to glycine or to other agonists, as this variant expands the potential heterogeneity among glycine receptors.     

Alanine-scanning mutagenesis in the signature disulfide loop of the glycine receptor alpha 1 subunit: critical residues for activation and modulation

The glycine receptor enables the generation of inhibitory postsynaptic currents at synapses via neurotransmitter-dependent activation. These receptors belong to the ligand-gated ion channel gene superfamily, in which all members are comprised of five subunits, each of which possesses a signature 13-residue disulfide loop (Cys loop) in the extracellular domain. In this study, we used alanine-scanning mutagenesis of the residues between C138 and C152 of the Cys loop of the glycine receptor alpha1 subunit to identify residues critical for receptor activation and allosteric modulation. Mutation of L142, F145, or P146 to alanine produced decreases in the potency, maximal amplitude, and Hill coefficient for currents elicited by glycine and impaired receptor activation by the agonist taurine. These residues, along with D148, are positionally conserved in the family of LGIC subunits. Mutation at several other positions had little or no effect. The inhaled anesthetics halothane and isoflurane potentiate submaximal agonist responses at wild-type receptors, via an allosteric site. The mutations L142A, F145A, P146A, and D148A abolished positive modulation by these anesthetics, in some cases revealing a small inhibitory effect. A molecular model of the glycine receptor alpha1 subunit suggests that the Cys loop is positioned in a region of the receptor at the interface between the extracellular and transmembrane domains and that the critical functional residues identified here lie along the face of a predominantly hydrophobic surface. The present data implicate the Cys loop as an important functional moiety in the process of glycine receptor activation and allosteric regulation by anesthetics.     

Role of the large cytoplasmic loop of the alpha 7 neuronal nicotinic acetylcholine receptor subunit in receptor expression and function

The role of the large intracellular loop of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in the expression of functional channels was studied. For this purpose, systematic deletions and substitutions were made throughout the loop and the ability of the mutated alpha7 subunits to support expression of functional nAChRs at the Xenopus oocyte membrane was tested. Surface nAChR expression was abolished upon removal of sequences at two regions, a 29-amino acid segment close to the N-terminus of the loop (amino acids 297-325) and adjacent to the third transmembrane region and an 11-amino acid segment near the fourth transmembrane region. Some residues (amino acids 317-322) within the 29 amino acids N-terminal segment could be substituted by others but not deleted without loss of expression, suggesting that a certain structure, determined by the number of amino acids rather than by their identity, has to be maintained in this region. The contiguous sequence M323 K324 R325 did not tolerate deletions and substitutions. Removal of the rest of the cytoplasmic loop was not deleterious; even higher expression levels (2-4-fold) were obtained upon large deletions of the loop (Delta399-432 and Delta339-370). High expression levels were observed provided that a minimal sequence of three amino acids (E371, G372, and M373) was present. In addition, some electrophysiological properties of mutant nAChRs were modified. Substitution of the EGM sequence by other protein segments produced a variety of effects, but, in general, insertions were not well tolerated, suggesting the existence of tight structural restrictions in the large cytoplasmic region of the rat alpha7 subunit.     

A highly conserved aspartic acid residue in the signature disulfide loop of the alpha 1 subunit is a determinant of gating in the glycine receptor

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission. This gene superfamily includes the nicotinic acetylcholine, GABAA/C, 5-hydroxytryptamine type 3, and glycine receptors. A signature disulfide loop (Cys loop) in the extracellular domain is a structural motif common to all LGIC member subunits. Here we report that a highly conserved aspartic acid residue within the Cys loop at position 148 (Asp-148) of the glycine receptor alpha1 subunit is critical in the process of receptor activation. Mutation of this acidic residue to the basic amino acid lysine produces a large decrease in the potency of glycine, produces a decrease in the Hill slope, and converts taurine from a full agonist to a partial agonist; these data are consistent with a molecular defect in the receptor gating mechanism. Additional mutation of Asp-148 shows that alterations in the EC50 for agonists are dependent upon the charge of the side chain at this position and not molecular volume, polarity, or hydropathy. This study implicates negative charge at position Asp-148 as a critical component of the process in which agonist binding is coupled to channel gating. This finding adds to an emerging body of evidence supporting the involvement of the Cys loop in the gating mechanism of the LGICs.     

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.     

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.     

Nmf11 is a novel ENU-induced mutation in the mouse glycine receptor alpha 1 subunit

Nmf11 is an N-ethyl-N-nitrosourea–induced recessive mouse mutation. In this article we show that the mutation is in the gene that encodes the glycine receptor alpha 1 subunit (Glra1). The new Glra1 mutation appears to affect glycine’s inhibitory neurotransmission in the central nervous system (CNS) of the nmf11 homozygotes, which suffer from a severe startle disease–related phenotype and die by postnatal day 21. The nmf11 mutation involves a C-to-A transition of nucleotide 518, which results in the N46K substitution in the long extracellular NH₂ terminal or ligand-binding domain of the GLRA1 mature protein. The mutation does not result in reduced expression of GLRA1 at the mRNA or protein levels and the mutant glycine receptor localizes properly in synaptic sites of nmf11 homozygotes.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

Agonist- and competitive antagonist-induced movement of loop 5 on the alpha subunit of the neuronal alpha4beta4 nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that rapidly convert a chemical signal into an electrical signal. Although the structure of the nAChR is quite well described, the coupling between agonist binding and channel gating is still under debate. In this study, we probed local conformational transitions on the neuronal α4β4 nAChR by specifically tethering a conformation-sensitive fluorescent dye on αG98C located on loop 5 (L5), and simultaneously monitoring fluorescence intensity and current after expression in Xenopus oocytes. The potency of acetylcholine (ACh) was significantly higher in the cysteine mutant and further increased upon tetramethylrhodamine-6-maleimide labeling, suggesting a role of L5 in binding or gating. Structural reorganizations of L5 were shown to occur upon activation, as revealed by the fluorescence intensity increase during ACh exposure. Fluorescence changes were also detected at ACh concentrations lower than needed for current activation, suggesting a movement of L5 for a closed, resting or desensitized state. The competitive antagonist dihydro-β-erythroidine also induced a movement of L5 although at concentrations significantly higher than needed for current inhibition. Consequently L5, located inside the lumen of the pentamer, plays a role in both activation and inhibition of the nAChR.     

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.     

Mechanisms of homomeric alpha1 glycine receptor endocytosis

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.     

Editing modifies the GABA(A) receptor subunit alpha3

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development.