Glenn H. Dillon

Sigma-1 Receptor Regulation of Voltage-Gated Calcium Channels Involves a Direct Interaction

PURPOSE The sigma-1 receptor belongs to a recently discovered family of transmembrane proteins expressed in the central nervous system, including the eye, and mediates the regulation of ion channels. The exact function of sigma receptors remains to be elucidated. The purpose of this study was to investigate the effect of sigma-1 receptor ligands on calcium homeostasis in a retinal ganglion cell line (RGC)-5 and rat primary RGCs. METHODS Calcium imaging was used to assess the effect of sigma-1 receptor agonist (+)-N-allylnormetazocine ((+)-SKF10047) on potassium chloride (KCl)-induced calcium influx in RGC-5. The whole-cell patch clamp technique was used to analyze the effect of (+)-SKF10047 on calcium currents in primary RGCs. Coimmunoprecipitation assessed the interaction between the sigma-1 receptor and the L-type voltage-gated calcium channel. RESULTS The sigma-1 receptor agonist (+)-SKF10047 inhibited potassium chloride (KCl)-induced calcium influx. The sigma-1 receptor antagonist, BD1047, reversed the inhibitory effect of (+)-SKF10047. Whole-cell patch clamp recordings of rat cultured primary RGCs demonstrated that (+)-SKF10047 inhibited calcium currents. Coimmunoprecipitation studies demonstrated an association between L-type calcium channels and the sigma-1 receptors. CONCLUSIONS These results suggest that sigma-1 receptor activation can regulate calcium homeostasis and signaling in RGCs, likely by directly influencing the activity of L-type voltage-gated calcium channels. Regulation of calcium influx in RGCs by sigma-1 receptor ligands may represent in part the neuroprotective effect of sigma-1 receptors.

PKC modulation of GABAA receptor endocytosis and function is inhibited by mutation of a dileucine motif within the receptor β2 subunit

The modulation of GABAA receptors by protein kinase C is complex and involves effects on both ion channel function and receptor trafficking. Although PKC regulates receptor cell surface expression the mechanism is not well understood. Using immunofluorescence studies in HEK 293 cells, we demonstrate that activation of PKC by the phorbol ester PMA promotes receptor endocytosis and is dependent on the presence of a gamma subunit. This endocytosis is blocked by the dominant negative dynamin mutant K44A indicating that PKC-induced receptor endocytosis involves the dynamin endocytic pathway. Mutation of a dileucine motif within the receptor beta2 subunit inhibits the effect of PKC activation on receptor endocytosis. Using patch clamp analysis, we show that PKC activation produces a robust inhibition of GABA-gated chloride currents in cells expressing wildtype GABAA receptors, but it is ineffective in modulating receptors lacking the dileucine motif. Furthermore, the introduction into the patch pipette of a 10-amino acid peptide corresponding to the dileucine motif present in the receptor beta2 subunit prevents PKC modulation of wildtype recombinant receptors. Furthermore, in cerebral cortical neuronal slices inclusion of this peptide in the patch pipette prevents PKC modulation of native GABAA receptors. Using limited chymotrypsin digestion assays, we also show that PKC increases receptor internalization in primary cultures of cerebral cortical neurons. Lastly, PKC inhibitors do not block constitutive receptor endocytosis or affect GABA-gated chloride currents suggesting that PKC-dependent phosphorylation is not required for GABAA receptor endocytosis but plays a modulatory role in the process.

The tyrosine kinase inhibitor genistein directly inhibits GABAA receptors

The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits GABAA receptors through a direct effect. Extracellular application of genistein and GABA reversibly inhibited GABA-activated currents recorded from HEK293 cells expressing rat alpha1beta2gamma2S or alpha1beta2 receptors, even when genistein was preequilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited GABA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the GABA response. Our results demonstrate that genistein has a direct inhibitory effect on GABAA receptors that is not mediated via inhibition of tyrosine kinase.

Inhibition of type a GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipines effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.

Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABAA receptors

We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1, 5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In alpha2beta2gamma2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl(-) current amplitude, and subsequently enhanced decay of GABA-activated Cl(-) currents. The half-maximal inhibitory concentration (IC(50)) for picrotoxin in alpha2beta2gamma2 receptors was 10.3+/-1.6 microM. The alpha subunit isoform did not affect picrotoxin-induced inhibition, as IC(50) values for alpha3beta2gamma2 (5.1+/-0.7 microM) and alpha6beta2gamma2 receptors (7.2+/-0.4 microM) were comparable to those obtained in alpha2beta2gamma2 receptors. Interestingly, in receptors lacking an alpha subunit (beta2gamma2 configuration), picrotoxin had a markedly lower IC(50) (0.5+/-0.05 microM) compared to alpha-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC(50) values were comparable in all alphabetagamma-containing receptors, but the IC(50) in beta2gamma2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in alpha2beta2gamma2 and beta2gamma2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxins ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an alpha subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor.

The GABAA receptor antagonist picrotoxin inhibits 5-hydroxytryptamine type 3A receptors

For a number of years it has been known that the CNS convulsant picrotoxin inhibits the GABA(A) receptor, an anion-selective member of the ligand-gated ion channel (LGIC) superfamily. PTX also inhibits other anion-selective LGIC members, such as GABA(C), glycine and glutamate-gated Cl(-) channels. In the present report, we tested the ability of picrotoxin to inhibit cation-selective 5-HT(3A) receptors. Murine 5-HT(3A) receptors were expressed in HEK293 cells, and functionally evaluated using whole-cell patch clamp recording. Picrotoxin inhibited 5-HT-gated currents in a concentration-dependent manner, with an IC(50) of approximately 30 microM. Moreover, the blockade by PTX was non-competitive and use-facilitated. Pentylenetetrazole and U-93631, ligands that act at a domain similar to that of picrotoxin in GABA(A) receptors, also inhibited the 5-HT(3A) receptor. For each ligand tested, its potency was 5-10 fold lower than typically observed in GABA(A) receptors. Our results demonstrate that, in addition to being a relatively non-selective inhibitor of anionic LGICs, picrotoxin also inhibits the cation-selective 5-HT(3A) receptor. Moreover, the fact that both PTZ and U-93631 similarly inhibit the 5-HT(3A) receptor is consistent with the suggestion that the site of picrotoxin action in this receptor may be comparable to that in anion-selective LGICs.

Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor.

Genistein, a tyrosine kinase inhibitor, has been widely used to examine potential effects of protein tyrosine kinase (PTK)-mediated regulation of receptor/channel function. Alteration of ion channel function in the presence of genistein has typically led to the conclusion that PTK regulates the activity of the channel under investigation. In the present report, we have assessed the possibility that genistein directly inhibits the glycine receptor, independent of effects on protein tyrosine kinase. Coapplication of genistein with glycine reversibly inhibited the strychnine-sensitive, glycine-activated current recorded from hypothalamic neurons. The time course of genistein action was rapid (within ms). Equilibration of genistein in the intracellular solution did not affect the ability of extracellularly applied genistein to inhibit the glycine response. Glycine concentration-response profiles generated in the absence and presence of genistein indicated the block was due to non-competitive antagonism. The genistein effect also displayed voltage-dependence. Daidzein, an analog of genistein that does not block protein kinases, also inhibited glycine-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the glycine response. Our results demonstrate that the tyrosine kinase inhibitor genistein has a direct inhibitory effect on glycine receptors that is not mediated via inhibition of PTK.

Carisoprodol-Mediated Modulation of GABAA Receptors: In Vitro and in Vivo Studies

Carisoprodol is a frequently prescribed muscle relaxant. In recent years, this drug has been increasingly abused. The effects of carisoprodol have been attributed to its metabolite, meprobamate, a controlled substance that produces sedation via GABAA receptors (GABAARs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABAAR function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human α1β2γ2 GABAAR function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric ρ1 GABA or glycine α1 receptors. In the absence of GABA, carisoprodol produced picrotoxin-sensitive, inward currents that were significantly larger than those produced by meprobamate, suggesting carisoprodol may directly produce GABAergic effects in vivo. When administered to mice via intraperitoneal or oral routes, carisoprodol elicited locomotor depression within 8 to 12 min after injection. Intraperitoneal administration of meprobamate depressed locomotor activity in the same time frame. In drug discrimination studies with carisoprodol-trained rats, the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate each substituted for carisoprodol in a dose-dependent manner. In accordance with findings in vitro, the discriminative stimulus effects of carisoprodol were antagonized by a barbiturate antagonist, bemegride, but not by the benzodiazepine site antagonist, flumazenil. The results of our studies in vivo and in vitro collectively suggest the barbiturate-like effects of carisoprodol may not be due solely to its metabolite, meprobamate. Furthermore, the functional traits we have identified probably contribute to the abuse potential of carisoprodol.

Comparison of the Binding and Functional Properties of Two Structurally Different D2 Dopamine Receptor Subtype Selective Compounds

We previously reported on the synthesis of substituted phenyl-4-hydroxy-1-piperidyl indole analogues with nanomolar affinity at D2 dopamine receptors, ranging from 10- to 100-fold selective for D2 compared to the D3 dopamine receptor subtype. More recently, we evaluated a panel of aripiprazole analogues, identifying several analogues that also exhibit D2 vs D3 dopamine receptor binding selectivity. These studies further characterize the intrinsic efficacy of the compound with the greatest binding selectivity from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled inward-rectifying potassium (GIRK) channel activation assay, and a cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a neutral antagonist at D2 dopamine receptors using all three assays. SV-III-130s is a partial agonist using an adenylyl cyclase inhibition assay but an antagonist in the GIRK and phospho ERK1/2 assays. To define the molecular basis for the binding selectivity, the affinity of these two compounds was evaluated using (a) wild type human D2 and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors. Computer-assisted modeling techniques were used to dock these compounds to the human D2 and D3 dopamine receptor subtypes. It is hoped that these studies on D2 receptor selective ligands will be useful in the future design of (a) receptor selective ligands used to define the function of D2-like receptor subtypes, (b) novel pharmacotherapeutic agents, and/or (c) in vitro and in vivo imaging agents.

Stoichiometric analysis of the TM2 6′ phenylalanine mutation on desensitization in α1β2 and α1β2γ2 GABAA receptors

Abstract The presence of phenylalanine (F) at the 6′ position of transmembrane domain 2 (TM2) in the α4 subunit of α4β2 nicotinic receptors enhances desensitization. As the GABA A receptor affords the ability to study the influence of as few as one and as many as five Fs at this position, we have used it to investigate potential subunit- and stoichiometry-dependent effects of the TM2 6′F mutation on desensitization. Whereas the presence of one F at this position decreased extent of desensitization, desensitization was increased in all configurations that included two or more Fs at the TM2 6′ position; desensitization was particularly rapid with 3 or 4 F residues present. Our results demonstrate the ability of F residues at the TM2 6′ position to modulate desensitization is likely conserved in the cys-loop family of ligand-gated ion channels. Moreover, our findings demonstrate both stoichiometric- and subunit-dependent effects of the ability of this mutation to regulate desensitization in GABA A receptors.