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PubMed1995

Molecular diversity of GABA-gated chloride channels in the rat anterior pituitary.

mRNA expression of GABA-gated Cl(-)-channels in rat antepituitary was evaluated by using an reverse-transcribed (RT)-polymerase chain reaction (RT-PCR) method with degenerate and specific oligonucleotides. The main result of our findings is that the antepituitary expresses mRNAs encoding alpha 4 and rho 1 GABA receptor subunits. These two subunits are believed to be, respectively, constituents of benzodiazepine-insensitive GABAA and GABAC receptors in the CNS. This molecular analysis is consistent with the pharmacological diversity of GABA receptors in pituitary cells.

Animals↗
PubMed2006

Crystal structure of the cytoplasmic domain of the chloride channel ClC-0.

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.

Amino Acid Sequence↗
PubMed1997

Probing the major skeletal muscle chloride channel with Zn2+ and other sulfhydryl-reactive compounds.

The sensitivity of the human skeletal muscle Cl- channel, hClC-1, towards various sulfhydryl-reactive agents was tested with the channel expressed in Xenopus oocytes and in human embryonic kidney cells. External but not internal Zn2+, at 1 mM, substantially reduced the current without affecting activation parameters. External Cd2+ and Hg2+ as well as organic mercurial compounds reduced the Cl- currents to a similar degree. With the mutant channel hClC-1 D136G, presumed to have a defective voltage sensor, external Zn2+ also reduced the current without effect on the altered gating. These findings suggest that hClC-1 contains cysteine residues near the extracellular face that may directly influence ion conduction. Since Zn2+ can also bind to histidine side chains, we tested the effect of compounds with either more cysteine- or more histidine-specificity. The results confirm the involvement of cysteine(s) in the observed effects but do not exclude the involvement of histidine(s).

Chloride Channels↗
PubMed1996

Inhibition of GABA-gated chloride channels in brain by the arachidonic acid metabolite, thromboxane A2.

Previously, we showed that arachidonic acid and prostaglandin metabolites inhibited GABAA responses in rat cerebral cortex. Thromboxane A2 (TXA2), a metabolite of arachidonic acid, has potent actions on blood vessels and platelets, but its actions on neurons are not known. Here, we examined the effects of several TXA2 analogs on the functional and binding characteristics of GABAA receptors in rat brain. The stable analogs of TXA2, pinane and carbocyclic TXA2, and the TXA2 agonist, U-46619, inhibited muscimol-induced 36Cl- uptake in cerebral cortical synaptoneurosomes. Carbocyclic TXA2 decreased the maximal response to muscimol, consistent with a non-competitive interaction. The TXA2 antagonist, SQ 25,548, did not block the effects of either arachidonic acid or carbocyclic TXA2. Neither the biologically inactive metabolite of TXA2, TXB2, nor carbacyclin, a stable analog of prostacyclin (prostaglandin I2) had an effect on GABAA responses. Thus the pharmacology differs from that in vascular smooth muscle and platelets. To determine if GABAA receptors were sensitive to the thromboxanes, the effect of pinane TXA2 on the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography. Pinane TXA2 inhibited [35S]TBPS binding in a regionally selective and non-competitive manner; the greatest inhibition was in the cerebral cortex, hippocampus and striatum, areas which are selectively vulnerable to cerebral ischemia. We conclude that TXA2 can interact with neuronal membranes to inhibit GABA receptor function, independent of its actions on the cerebrovasculature and on glial cells. This may be important during pathologic states such as ischemia, when TXA2 accumulates in extracellular spaces.

Animals↗
PubMed2004

Novel mutations of the chloride channel Kb gene in two Japanese patients clinically diagnosed as Bartter syndrome with hypocalciuria.

Hypokalemic metabolic tubulopathy, such as in Bartter syndrome and Gitelman syndrome, is caused by the dysfunction of renal electrolyte transporters. Despite advances in molecular genetics with regard to hypokalemic metabolic tubulopathy, recent reports have suggested that the phenotype-genotype correlation is still confusing, especially in classic Bartter and Gitelman syndromes. We report here two Japanese patients who suffered from clinically diagnosed classic Bartter syndrome but who presented hypocalciuria. Hypocalciuria is generally believed to be a pathognomonic finding of NCCT malfunction. To better understand the genotype-phenotype correlation in these two cases, we screened four renal electrolyte transporter genes [Na-K-2Cl cotransporter (NKCC2), renal outer medullary K channel (ROMK), Cl channel Kb (ClC-Kb), and Na-Cl cotransporter (NCCT)] by the PCR direct sequencing method. We identified three ClC-Kb allelic variants, including two new mutations (L27R and W610X in patient 1 and a G to C substitution of a 3' splice site of intron 2 and W610X in patient 2). We did not find any mutations in the other three genes. Our present data suggest that some ClC-Kb mutations may affect calcium handling in renal tubular cells.

Adolescent↗
PubMed1996

Extracellular acidosis and chloride channel inhibitors act in the late phase of cellular injury to prevent death.

Extracellular acidosis is cytoprotective in several models against anoxia/hypoxia and a variety of toxicants. The goal of this study was to determine the temporal relationships among toxicant exposure, the initiation of extracellular acidosis, Cl. Influx, Cl- channel inhibition and the onset of cellular death in rabbit renal proximal tubule suspensions. Extracellular acidosis was produced by adding HCl or H2SO4 to renal proximal tubule suspensions to decrease the extracellular buffer pH to 6.4 or by resuspending renal proximal tubules in a pH 6.4 buffer. The initiation of extracellular acidosis 15 min after the mitochondrial inhibitors antimycin A or carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone addition, a time point in which adenosine triphosphate levels are depleted and intracellular K+ is decreased, ameliorated lactate dehydrogenase release, a marker of necrotic cellular death. The initiation of extracellular acidosis 120 min after the addition of the toxicants tetrafluoroethyl-L-cysteine or t-butyl hydroperoxide decreased lactate dehydrogenase release 120 min later. Increased Cl- influx is an important step during the late phase of toxicant-induced cellular injury. Therefore, we determined if extracellular acidosis cytoprotection was associated with inhibition of Cl- influx and whether the Cl- channel inhibitors indanyloxyacetic acid (1.0 mM). niflumic acid (100 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (100 microM) decreased Cl- influx and cellular death in renal proximal tubules exposed to antimycin A. Indeed, all three Cl- channel inhibitors significantly decreased 38Cl- influx and cellular death. In contrast, extracellular acidosis did not decrease 38Cl- influx but did prevent lactate dehydrogenase release. These results demonstrate that extracellular acidosis cytoprotection occurs during the late phase of cellular injury at a site distal to Cl- influx. Furthermore, the Cl- influx that occurs during the late phase of cellular injury and is critical for cellular swelling and lysis is sensitive to 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, niflumic acid and indanyloxyacetic acid.

Acidosis↗
PubMed2005

Role of chloride channels in bradykinin-induced guinea pig airway vagal C-fibre activation.

We tested the hypothesis that an ionic current carried by chloride ions contributes to bradykinin (BK)-induced membrane depolarization and activation of vagal afferent C-fibres. In an ex vivo innervated trachea/bronchus preparation, BK (1 microM) consistently produced action potential discharge in vagal afferent C-fibres with receptive fields in the trachea or main stem bronchus. The Ca2+-activated Cl- channel (CLCA) inhibitor, niflumic acid (NFA, 100 microM), significantly reduced BK-induced action potential discharge to 21 +/- 7% of the control BK response. NFA did not inhibit capsaicin-induced or citric-acid-induced action potential discharge in tracheal C-fibres. The inhibitory effect of NFA was mimicked by another CLCA inhibitor, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 microM). NFA also inhibited the BK-induced inward current in gramicidin-perforated whole-cell patch-clamp recordings of capsaicin-sensitive jugular ganglion neurones retrogradely labelled from the airways. NFA did not inhibit the BK-induced increase in intracellular free Ca2+. The TRPV1 inhibitor, iodo-resiniferatoxin (1 microM), also partially inhibited BK-induced action potential discharge, and the combination of iodo-resiniferatoxin and NFA virtually abolished the BK-induced action potential discharge. We concluded that in vagal afferent C-fibres, BK evokes membrane depolarization and action potential discharge through the additive effects of TRPV1 and Cl- channel activation.

Afferent Pathways↗
PubMed1995

CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-.

Adenosine Triphosphate↗
PubMed1998

Calcium-inhibitable current in cultured embryonic chick cardiac myocytes: possibly via a novel chloride channel.

The role of extracellular Ca2+ (Ca(2+)o) in the modulation of cardiac Cl- currents (I(Cl)) such as those activated by cAMP or swelling is uncertain. The effects of Ca(2+)o and extracellular cadmium (Cd(2+)o) on Cl- currents in cultured chick cardiac myocytes were investigated in Na+- and K+-free internal and external solutions using the whole-cell patch-clamp technique. In the absence of Na+ and K+ internally and externally, the whole-cell current was predominantly I(Cl). In the absence of cAMP, removal of Ca(2+)o (+ 1 mM EGTA) resulted in an increase in the current that was suppressed by reduction of Cl(o)- with a rightward shift of the zero-current potential towards the CI- reversal potential. We designated this current as a Ca2+-inhibitable I(Cl). Addition of 0.5 mM Cd(2+)o with or without removal of Ca(2+)o also resulted in a 1.5- to 2.0-fold increase in I(Cl) that was attenuated by 1 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). Under similar conditions, I(Cl) activated by Cd(2+)o (in 1 mM Ca(2+)o solution) was not further increased by subsequent removal of Ca(2+)o, suggesting that addition of Cd(2+)o and removal of Ca(2+)o activated the same I(Cl). In contrast, exposure to 1 microM forskolin further enhanced I(Cl) in the presence of Cd(2+)o. With 10 microM cAMP in the pipette solution, Ca2+-inhibitable I(Cl) could be activated in myocytes that do not possess cAMP-activated Cl- channels, indicating that activation of Ca2+-inhibitable I(Cl) does not require cAMP. In the presence of cAMP, in cells that display the cAMP-activated I(Cl), removal of Ca(2+)o resulted in a further increase in I(Cl) comparable to the Ca2+-inhibitable I(Cl). The Ca2+-inhibitable I(Cl) was minimized when pipette solutions contained 1.5 microM Ca2+. These results suggest that removal of Ca(2+)o or application of Cd(2+)o activates a Ca2+-inhibitable I(Cl) that is distinct from the cAMP-activated I(Cl).

Animals↗
PubMed1998

Activation of bovine tracheal chloride channels by amino group-specific reagents.

1. The predominant Cl- channel in bovine tracheal epithelial cells has a conductance of approximately 71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein-modifying reagents on channel function and found that amino groups are critically involved in gating. 2. Patch clamp studies showed that lysine-specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway. 3. Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time. 4. Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site-directed mutagenesis and pharmacological activation of Cl- channels in epithelial cells.

Animals↗
PubMed1997

Independent gating of single pores in CLC-0 chloride channels.

The Cl- channel from the Torpedo electric organ, CLC-0, is the prototype of a large gene family of Cl- channels. At the single-channel level, CLC-0 shows a "double-barreled" behavior. Recently it was shown that CLC-0 is a dimer, and it was suggested that each subunit forms a single pore. The two protopores are gated individually by a fast voltage and anion-dependent gating mechanism. A slower common gating mechanism operates on both pores simultaneously. Previously, wild-type/mutant heteromeric channels had been constructed that display a large wild-type pore and small mutant pore. Here we use patch-clamp recording of single wild-type and mutant CLC-0 channels to investigate in detail the dependence of the gating of one protopore on the physically attached neighboring pore. No difference in rate constants of opening and closing of protopores could be found comparing homomeric wild-type and heteromeric wild-type/mutant channels. In addition, detailed kinetic analysis reveals that gating of single subunits is not correlated with the gating of the neighboring subunit. The results are consistent with the view that permeation and fast gating of individual pores are fully independent of the neighboring pore. Because the two subunits are associated in a common protein complex, opening and closing transitions of individual pores are probably due to only small conformational changes in each pore. In addition to the fast and slow gating mechanisms known previously for CLC-0, in the course of this study we occasionally observed an additional gating process that led to relatively long closures of single pores.

Animals↗
PubMed2006

Similar chloride channels in the connecting tubule and cortical collecting duct of the mouse kidney.

Using the patch-clamp technique, we investigated Cl- channels on the basolateral membrane of the connecting tubule (CNT) and cortical collecting duct (CCD). We found a approximately 10-pS channel in CNT cell-attached patches. Substitution of sodium gluconate for NaCl in the pipette shifted the reversal potential by +25 mV, whereas N-methyl-D-gluconate chloride had no effect, indicating anion selectivity. On inside-out patches, we determined a selectivity sequence of Cl- > Br- approximately NO3(-) > F-, which is compatible with that of ClC-K2, a Cl- channel in the distal nephron. In addition, the number of open channels (NP(o)) measured in cell-attached patches was significantly increased when Ca2+ concentration or pH in the pipette was increased, which is another characteristic of ClC-K. These findings suggest that the basis for this channel is ClC-K2. A similar Cl- channel was found in CCD patches. Because CNT and CCD are heterogeneous tissues, we studied the cellular distribution of the Cl- channel using recording conditions (KCl-rich solution in the pipette) that allowed us to detect simultaneously Cl- channels and inwardly rectifying K+ channels. We detected Cl- channels alone in 45% and 42% and K+ channels alone in 51% and 58% of CNT and CCD patches, respectively. Cl- and K+ channels were recorded simultaneously from two patches (4% of patches) in the CNT and from none of the patches in the CCD. This indicates that Cl- and K+ channels are located in different cell types, which we suggest may be the intercalated cells and principal cells, respectively.

Animals↗
PubMed2003

A novel mutation in the chloride channel gene, CLCNKB, as a cause of Gitelman and Bartter syndromes.

BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary hypokalemic tubulopathies with distinct phenotypic features. GS has been considered a genetically homogeneous disorder caused by mutation in the gene encoding the NaCl cotransporter (TSC) of the distal convoluted tubule. In contrast, BS is caused by mutations in the genes encoding either the Na-K-2Cl cotransporter (NKCC2), the K+ channel (ROMK) or the Cl- channel (ClC-Kb) of the thick ascending limb. The purpose of this study was to examine the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel with hereditary hypokalemic tubulopathy. METHODS: Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent genetic linkage analysis. Mutation analysis was performed in affected individuals. RESULTS: Evaluation of affected family members (age range 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS included late age of presentation (>15 years) in 7 patients (58%), normal growth in 9 (75%), hypomagnesemia (SMg <0.7mmol/L) in 5 (42%), hypermagnesiuria (FEMg>5%) in 6 (50%) and hypocalciuria (urinary calcium/creatinine mmol/mmol <0.15) in 5 (42%). Features typical of CBS included early age of presentation (<1 year) in 3 (25%), polyuria/dehydration in 4 (33%), growth retardation in 3 (25%), hypercalciuria (urinary calcium/creatinine mmol/mmoverline>0.55) in 4 (33%) and nephrolithiasis in 1 (8%). Linkage analysis in affected patients excluded the TSC gene, SLC12A3, as the mutated gene, but demonstrated linkage to the Cl- channel gene, CLCNKB, on chromosome 1p36. Mutation analysis by direct sequencing revealed a novel homozygous missense mutation, arginine 438 to histidine (R438H), in exon 13 of CLCNKB in all patients. A restriction fragment length polymorphism (RFLP) analysis has been developed to aid in genotyping of family members. CONCLUSIONS: Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.

Adult↗
PubMed1997

Modulation of CFTR chloride channels by calyculin A and genistein.

Modulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by calyculin A and genistein was studied in Hi-5 insect cells infected with baculovirus containing the wild-type CFTR cDNA. In cell-attached patches, CFTR channel activity was not observed until stimulated by forskolin in 90% of the cells, suggesting a low level of basal adenosine 3',5'-cyclic monophosphate activity. Calyculin A, a specific inhibitor of phosphatases 1 and 2A, increased forskolin-induced CFTR activity by 17.2-fold. CFTR channel currents did not deactivate completely after forskolin was withdrawn in the continued presence of calyculin A. Genistein enhanced forskolin-induced CFTR activity by 44.9-fold but could neither activate the CFTR by itself nor prevent complete deactivation on removal of forskolin. Genistein together with calyculin A could adequately prevent deactivation of CFTR currents. Noise analysis of the macroscopic CFTR currents revealed significant differences in the mean current-variance-relationship and the corner frequency of the noise spectra between currents activated by forskolin plus genistein and those activated by forskolin plus calyculin A. Furthermore, genistein enhanced CFTR activity induced by saturating concentrations of forskolin and calyculin A. Our results suggest that genistein and calyculin A modulate the CFTR by different mechanisms and that genistein might inhibit calyculin A-insensitive dephosphorylation of the CFTR.

3T3 Cells↗
PubMed2001

Different fast-gate regulation by external Cl(-) and H(+) of the muscle-type ClC chloride channels.

The fast gate of the muscle-type ClC channels (ClC-0 and ClC-1) opens in response to the change of membrane potential (V). This gating process is intimately associated with the binding of external Cl(-) to the channel pore in a way that the occupancy of Cl(-) on the binding site increases the channel's open probability (P(o)). External H(+) also enhances the fast-gate opening in these channels, prompting a hypothesis that protonation of the binding site may increase the Cl(-) binding affinity, and this is possibly the underlying mechanism for the H(+) modulation. However, Cl(-) and H(+), modulate the fast-gate P(o)-V curve in different ways. Varying the external Cl(-) concentrations ([Cl(-)](o)) shifts the P(o)-V curve in parallel along the voltage axis, whereas reducing external pH mainly increases the minimal P(o) of the curve. Furthermore, H(+) modulations at saturating and nonsaturating [Cl(-)](o) are similar. Thus, the H(+) effect on the fast gating appears not to be a consequence of an increase in the Cl(-) binding affinity. We previously found that a hyperpolarization-favored opening process is important to determine the fast-gate P(o) of ClC-0 at very negative voltages. This [Cl(-)](o)-independent mechanism attracted little attention, but it appears to be the opening process that is modulated by external H(+).

Animals↗
PubMed2001

Tissue distribution and subcellular localization of the ClC-5 chloride channel in rat intestinal cells.

ClC-5 is the Cl- channel that is mutated in Dent's disease, an X-chromosome-linked disease characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. It is predominantly expressed in endocytically active renal proximal cells. We investigated whether this Cl- channel could also be expressed in intestinal tissues that have endocytotic machinery. ClC-5 mRNA was detected in the rat duodenum, jejunum, ileum, and colon. Western blot analyses revealed the presence of the 83-kDa ClC-5 protein in these tissues. Indirect immunofluorescence studies showed that ClC-5 was mainly concentrated in the cytoplasm above the nuclei of enterocytes and colon cells. ClC-5 partially colocalized with the transcytosed polymeric immunoglobulin receptor but was not detectable together with the brush-border-anchored sucrase isomaltase. A subfractionation of vesicles obtained by differential centrifugation showed that ClC-5 is associated with the vacuolar 70-kDa H+-ATPase and the small GTPases rab4 and rab5a, two markers of early endosomes. Thus these results indicate that ClC-5 is present in the small intestine and colon of rats and suggest that it plays a role in the endocytotic pathways of intestinal cells.

Animals↗
PubMedDate not supplied

Comparative photoaffinity labeling study between azidophenyl, difluoroazidophenyl, and tetrafluoroazidophenyl derivatives for the GABA-gated chloride channels.

Syntheses of 2-aryl-substituted photoactivatable derivatives of 5-tert-butyl-1,3-dithiane and their oxidized bis-sulfone are described. The 4-azidoaryl and the 3,5-difluoro-4-azidoaryl groups were chosen as photosensitive moieties. Azidoaryl derivatives 3 and 13 were synthesized by diazotization and azidation of their corresponding arylamine precursors. The o-difluoroazidophenyl derivatives 2 and 9 were synthesized by transformation of the o-difluoro-substituted lithiophenyl into the corresponding azido derivative. The reversible binding properties of the photosensitive probes were established on bovine cortex P2 membranes by displacement of [3H]-1-phenyl-4-tert-butyl- 2,6,7-trioxabicyclo[2.2.2]-octane ([3H]TBOB), a specific ligand for the channel blocker binding site. The 2-(3',5'-difluro-4'-azidophenyl)-5-tert-butyl- 1,3-dithianebis-sulfone, compound 2, exhibited the best Ki of about 11 nM, compared to Kis of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorophenyl analogue) and compared to Kis of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorophenyl analogue) and 3 (azidphenyl analogue). On irradiation, probe 2 (0.6 microM) produced 18% irreversible loss of TBOB binding sites in brain membranes while probe 3 did not produce any photoinactivation. The loss observed with 2 was fully protectable by TBOB, demonstrating the specificity of the photochemical inactivation by compound 2 for the convulsant site of the GABAA receptor. These results, when compared to the photoaffinity labeling results obtained with the tetrafluorinated probe 1 (25% selective irreversible photochemical inactivation), establish a hierarchy between fluorinated and nonfluorinated arylazido probes and strengthen the potential of the newly described difluorinated probe 2, combining high affinity and good labeling efficacy.

Affinity Labels↗
PubMed1998

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways.

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [3H]strychnine binding assays. In the wild-type (WT) GlyR, 1 microM 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.

Cell Line↗