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The purpose of this article is to summarize disparities in blood pressure (BP) by race in the United States, discuss evidence-based strategies to increase equity in BP, review recent American Heart Association BP equity initiatives, and highlight missed opportunities for achieving equity in hypertension. Over 122 million American adults have hypertension, with the highest prevalence among Black Americans. Racial disparities in hypertension and BP control in the United States are estimated to be the single largest contributor to the excess risk for cardiovascular disease among Black versus White adults. Worsening disparities in cardiovascular disease and life expectancy during the COVID-19 pandemic warrant an evaluation of the strategies and opportunities to increase equity in BP in the United States. Racial disparities in hypertension are largely driven by systemic inequities that limit access to quality education, economic opportunities, neighborhoods, and health care. To address these root causes, recent studies have evaluated evidence-based strategies, including community health workers, digital health interventions, team-based care, and mobile health care to enhance access to health education, screenings, and BP care in Black communities. In 2021, the American Heart Association made a $100 million pledge and 10 commitments to support health equity. This commitment included implementing multifaceted interventions with a focus on hypertension as a seminal risk factor contributing to disparities in cardiovascular disease mortality and morbidity. The American Heart Association is one organizational example of advocacy for equity in BP. Achieving equity nationwide will require sustained collaboration among individual stakeholders and public, private, and community organizations to address barriers across multiple socioecological levels., Competing Interests: V. Fontil is a recipient of a Small Business Innovation Research grant to commercialize a clinical decision-support tool for hypertension (Engage Rx) that he developed in his research work. G.H. Dillon is an employee of the American Heart Association. The other authors report no conflicts.

The coronavirus disease 2019 (COVID-19) pandemic has had worldwide repercussions for health care and research. In spring 2020, most non-COVID-19 research was halted, hindering research across the spectrum from laboratory-based experimental science to clinical research. Through the second half of 2020 and the first half of 2021, biomedical research, including cardiovascular science, only gradually restarted, with many restrictions on onsite activities, limited clinical research participation, and the challenges associated with working from home and caregiver responsibilities. Compounding these impediments, much of the global biomedical research infrastructure was redirected toward vaccine testing and deployment. This redirection of supply chains, personnel, and equipment has additionally hampered restoration of normal research activity. Transition to virtual interactions offset some of these limitations but did not adequately replace the need for scientific exchange and collaboration. Here, we outline key steps to reinvigorate biomedical research, including a call for increased support from the National Institutes of Health. We also call on academic institutions, publishers, reviewers, and supervisors to consider the impact of COVID-19 when assessing productivity, recognizing that the pandemic did not affect all equally. We identify trainees and junior investigators, especially those with caregiving roles, as most at risk of being lost from the biomedical workforce and identify steps to reduce the loss of these key investigators. Although the global pandemic highlighted the power of biomedical science to define, treat, and protect against threats to human health, significant investment in the biomedical workforce is required to maintain and promote well-being.

The muscle relaxant carisoprodol has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures, and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the type A GABA (GABA A ) receptor. These actions are subunit-dependent; compared with other GABA A receptors, carisoprodol has nominal direct gating effects in α 3 β 2 γ 2 receptors. Here, using site-directed mutagenesis and whole-cell patch-clamp electrophysiology in transiently transfected human embryonic kidney 293 cells, we examined the role of GABA A receptor α subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol. Mutation of α 3 valine at position 440 to leucine (present in the equivalent position in the α 1 subunit) significantly increased the direct gating effects of carisoprodol without affecting its allosteric modulatory effects. The corresponding reverse mutation, α 1(L415V), decreased carisoprodol direct gating potency and efficacy. Analysis of a series of amino acid mutations at the 415 position demonstrated that amino acid volume correlated positively with carisoprodol efficacy, whereas polarity inversely correlated with carisoprodol efficacy. We conclude that α 1(415) of TM4 is involved in the direct gating, but not allosteric modulatory, actions of carisoprodol. In addition, the orientation of alkyl or hydroxyl groups at this position influences direct gating effects. These findings support the likelihood that the direct gating and allosteric modulatory effects of carisoprodol are mediated via distinct binding sites., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Efavirenz is a widely prescribed medicine used to treat type 1 human immunodeficiency virus (HIV-1), the most prevalent pathogenic strain of the virus responsible for the acquired immune deficiency syndrome (AIDS) pandemic. Under prescribed dosing conditions, either alone or in combination therapy, efavirenz-induced CNS disturbances are frequently reported. Efavirenz was recently reported to interact in a similar concentration range with a number of receptors, transporters and ion channels including recombinant rat α1β2γ2 GABA A receptors whose actions were potentiated (Gatch et al., 2013; Dalwadi et al., 2016). Now we report on the molecular mechanism of efavirenz on GABA A receptors as a function of concentration and subunit composition via whole-cell recordings of GABA-activated currents from HEK293 cells expressing varying subunit configurations of GABA A receptors. Efavirenz elicited dual effects on the GABA response; it allosterically potentiated currents at low concentrations, whereas it inhibited currents at higher concentrations. The allosteric potentiating action on GABA A receptors was pronounced in the α1β2γ2, α2β2γ2 and α4β2γ2 configurations, greatly diminished in the α6β2γ2 configuration, and completely absent in the α3β2γ2 or α5β2γ2 configuration. In stark contrast, the inhibitory modulation of efavirenz at higher concentrations was evident in all subunit configurations examined. Moreover, efavirenz-induced modulatory effects were dependent on GABA concentration ([GABA]), with a pronounced impact on currents activated by low [GABA] but little effect at saturating [GABA]. Mutation of a highly-conserved threonine to phenylalanine in transmembrane domain 2 of the α1 subunit abolished the inhibitory effect of efavirenz in α1β2 receptors. Finally, mutations of any of the three conserved extracellular residues in α1/2/4 subunits to the conserved residues at the corresponding positions in α3/5 subunits (i.e., R84P, M89L or I120L) completely eliminated the potentiating effect of efavirenz in α1β2γ2 configuration. These findings demonstrate that efavirenz's positive allosteric modulation of the GABA A receptor is mediated via a novel allosteric site associated with the extracellular domain of the receptor., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABA A receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABA A receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 μM, and it had an IC 50 of 31 μM in human α1β2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the β2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABA A receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1β2 receptors to MB was similar to that observed in α1β2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn 2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and β2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABA A receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABA A receptor function, which may underlie some of the effects of MB on the CNS., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxβzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1β1γ2 and α1β2γ2 receptors, whereas allosteric effects were enhanced in α1β2 compared to α1β2γ2 receptors. In "extrasynaptic" (α1β3δ and α4β3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1-6 and z = 1-3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vascular dementia ranks as the second leading cause of dementia in the United States. However, its underlying pathophysiological mechanism is not fully understood and no effective treatment is available. The purpose of the current study was to evaluate long-term cognitive deficits induced by transient middle cerebral artery occlusion (tMCAO) in rats and to investigate the underlying mechanism. Sprague-Dawley rats were subjected to tMCAO or sham surgery. Behavior tests for locomotor activity and cognitive function were conducted at 7 or 30days after stroke. Hippocampal long term potentiation (LTP) and involvement of GABAergic neurotransmission were evaluated at 30days after sham surgery or stroke. Immunohistochemistry and Western blot analyses were conducted to determine the effect of tMCAO on cell signaling in the hippocampus. Transient MCAO induced a progressive deficiency in spatial performance. At 30days after stroke, no neuron loss or synaptic marker change in the hippocampus were observed. LTP in both hippocampi was reduced at 30days after stroke. This LTP impairment was prevented by blocking GABAA receptors. In addition, ERK activity was significantly reduced in both hippocampi. In summary, we identified a progressive decline in spatial learning and memory after ischemic stroke that correlates with suppression of hippocampal LTP, elevation of GABAergic neurotransmission, and inhibition of ERK activation. Our results indicate that the attenuation of GABAergic activity or enhancement of ERK/MAPK activation in the hippocampus might be potential therapeutic approaches to prevent or attenuate cognitive impairment after ischemic stroke., (© 2013.)

Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABAA and 5-HT(2A) receptors. In rodents, interaction with the 5-HT(2A) receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT(2A) receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT(2A)-knockout, mice. Despite having GABAA-potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that self-administer cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT(2A) receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication.

SV 293 [1-([5-methoxy-1H-indol-3-yl]methyl)-4-(4-[methylthio]​phenyl)piperidin-4-ol] binds with 100-fold higher affinity to human D2 receptors compared to the human D3 and D4 dopamine receptor subtypes. We investigated the intrinsic efficacy of this compound at the D2 dopamine receptor subtype using both: (1) a forskolin-dependent adenylyl cyclase inhibition assay and (2) an electrophysiological assay for evaluating coupling to G-protein-coupled inwardly rectifying potassium channels. In both assays SV 293 was found to be a neutral antagonist capable of blocking the effects of the full D2-like receptor agonist quinpirole. Based upon these results we propose that SV 293 is a useful pharmacological tool that can be used for both in vitro and in vivo studies to investigate the role of D2-like dopamine receptor subtypes in neurological, neuropsychiatric and movement disorders where dopaminergic pathways have been implicated., (Copyright © 2013 S. Karger AG, Basel.)

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.

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 N-methyl-d-aspartate (NMDA) receptors through a direct effect. Whole-cell NMDA-activated current was recorded in native receptors from mouse hippocampal slice culture and rat recombinant NR1aNR2A and NR1aNR2B receptors transiently expressed in HEK293 cells. Extracellular application of genistein and NMDA reversibly inhibited NMDA-activated current. The inhibition of NMDA-activated current by genistein applied externally was not affected when genistein was also pre-equilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited NMDA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the NMDA response. Moreover, genistein-induced inhibition of NMDA-activated current displayed concentration- and voltage-dependence. Our results demonstrate that genistein has a direct inhibitory effect on NMDA receptors that is not mediated via inhibition of tyrosine kinase. Thus, other PTK inhibitors may be more suitable for studying involvement of PTKs in NMDA receptor-mediated events., (Published by Elsevier Ltd.)

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 GABA(A) receptors (GABA(A)Rs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABA(A)R function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human alpha1beta2gamma2 GABA(A)R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric rho1 GABA or glycine alpha1 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.

Soma(®) (carisoprodol) is an increasingly abused, centrally-acting muscle relaxant. Despite the prevalence of carisoprodol abuse, its mechanism of action remains unclear. Its sedative effects, which contribute to its therapeutic and recreational use, are generally attributed to the actions of its primary metabolite, meprobamate, at GABA(A) receptors (GABA(A)R). Meprobamate is a controlled substance at the federal level; ironically, carisoprodol is not currently classified as such. Using behavioral and molecular pharmacological approaches, we recently demonstrated carisoprodol, itself, is capable of modulating GABA(A)R function in a manner similar to central nervous system depressants. Its functional similarities with this highly addictive class of drugs may contribute to the abuse potential of carisoprodol. The site of action of carisoprodol has not been identified; based on our studies, interaction with benzodiazepine or barbiturate sites is unlikely. These recent findings, when coupled with numerous reports in the literature, support the contention that the non-controlled status of carisoprodol should be reevaluated.

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.

L-type voltage-gated Ca(2+)channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces Ca(2+) influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca(2+) influx is not clearly understood. We show by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridine-insensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation.

The presence of phenylalanine (F) at the 6' position of transmembrane domain 2 (TM2) in the alpha4 subunit of alpha4beta2 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.

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.

The GABAA receptor is a ligand-gated ion channel whose function and activity can be regulated by ligand binding or alternatively may be influenced indirectly through the phosphorylation of specific subunits that comprise the GABAA receptor pentamer. With respect to phosphorylation, most studies have focused on either beta or gamma subunits, whereas the role of the alpha subunit as a relevant target of signaling kinases is largely unknown. Interestingly, we found a putative phosphorylation site for extracellular-signal regulated kinase (ERK), a key effector of the MAPK pathway, in almost all known alpha subunits of the GABAA receptor, including the ubiquitously expressed alpha1 subunit. To determine whether this putative ERK phosphorylation site was functionally relevant, we evaluated if ERK inhibition (through pharmacological inhibition of its upstream kinase, MEK) altered GABA-gated currents. Using HEK293 cells stably transfected with the alpha1beta2gamma2 form of the GABAA receptor, we found that UO126 reduced basal ERK phosphorylation and resulted in an enhancement of GABA-induced peak current amplitudes. Further, the enhancement of GABA-gated currents required an intact intracellular environment as it was robust in perforated patch recordings (which preserves the intracellular milieu), but absent in conventional whole-cell recordings (which dialyzes the cytosolic contents), supporting the involvement of an intracellular signaling pathway. Finally, mutation of the ERK phosphorylation site (T375-->A) prevented the UO126-induced enhancement of GABA-gated currents. Collectively, our results implicate the MAPK pathway as a negative modulator of GABAA receptor function, whose influence on GABA-gated currents may be mediated by phosphorylation of the alpha subunit., (Copyright 2006 Wiley Periodicals, Inc.)

Endogenous divalent cations Cu2+ and Zn2+ suppress the activity of glycine receptors (glyRs). Whereas residues critical for the effects of Zn2+ on glyRs have been identified, little is known about the determinants of Cu2+-mediated inhibition. In the present studies, we have assessed the potential commonality of Zn2+ and Cu2+-mediated inhibition of glyRs. Cu2+ potently inhibited recombinant human glycine alpha1 receptors, with an IC50 of 4.1+/-0.7 microM. Systematic mutation of extracellular histidine residues revealed that mutation H215A greatly reduced the inhibitory modulation by Cu2+. Substitution of H215 with C produced receptors with Cu2+ sensitivity similar to the wild type. Furthermore, modification of H215C with a thio-specific reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), reduced Cu2+ sensitivity of H215C receptors. However, mutation of other extracellular histidine residues including H107 and H109, which are known inhibitory Zn2+coordination sites, failed to influence inhibition of glycine currents by Cu2+. Moreover, mutation to alanine of two threonine residues (T112, T133) critical for Zn2+ inhibition had no effect (T133A) or only partial inhibitory effects (T112A) on Cu2+-induced inhibition. The double mutation, T112A/H215A, caused greater effects on Cu2+-mediated inhibition than either mutation alone. In addition, the glycine currents recorded from T112A/H215A mutant receptors were significantly potentiated by low concentrations of Cu2+. Our results have identified critical determinants of Cu2+-mediated inhibition of glyRs. Moreover, we demonstrate for the first time a clear difference in residues responsible for Cu2+-mediated compared to Zn2+-mediated inhibition of glyRs.

Amphibian oocyte and mammalian heterologous expression systems are often used to investigate the function of recombinant ion channels using electrophysiological techniques. Although both systems have yielded important information, the results obtained in these systems are sometimes conflicting. Oocytes and mammalian cells differ in their physiological temperature requirements. While room temperature is within the physiological temperature range for oocytes, this temperature is far below that required by mammalian cells. Since electrophysiological studies are often performed in both oocytes and mammalian cells at room temperature, we sought to determine if recording temperature could be a factor in some disparate results obtained in these cell types. For these studies, we examined phorbol ester modulation of GABA(A) and glycine receptors. Consistent with the literature, at room temperature, PMA (phorbol 12-myristate 13-acetate) produced a large reproducible decrease in the peak amplitude of GABA and glycine-gated currents in Xenopus oocytes. In contrast, PMA was ineffective in modulating these heterologously expressed receptors at room temperature in human embryonic kidney (HEK) 293 cells. However, when electrophysiological experiments were performed at 35 degrees C in HEK 293 cells, PMA decreased the function of these receptors. Our results indicate that the temperature at which electrophysiological studies are conducted is an important experimental variable. To determine the extent to which electrophysiological recordings are performed at physiological temperatures in HEK 293 cells, a PubMed search was conducted using the search terms "patch clamp" and "HEK" for the years 2003-2004. This search revealed that only 15% of the patch clamp studies were reported to have been conducted in the temperature range of 32-37 degrees C. The results of our study indicate that temperature is an important experimental variable that requires rational consideration in the design of electrophysiological experiments.

Allosteric modulation of mouse 5-Hydroxytryptamine(3A) (5-HT(3A)) and 5-HT(3A/B) receptor function by ethanol and trichloroethanol (TCEt) was assessed in HEK293 cells with whole cell patch-clamp electrophysiological recordings. Ethanol enhanced 5-HT(3A) receptor function, but had no effect on mouse 5-HT(3A/B) receptor mediated currents. The enhancing action of trichloroethanol (TCEt) on mouse 5-HT(3A/B) receptor function was much less than that observed in the mouse 5-HT(3A) receptor. Where alcohol-induced increases in peak amplitude were observed, the slope of the 20-80% rising phase of current onset was also enhanced, suggesting that increases in activation rate may be one mechanism through which alcohols enhance function of the 5-HT(3) receptors.

Previously, we reported that the GABA(A) receptor antagonist picrotoxin also antagonizes serotonin (5-HT)3 receptors and that its effects are subunit-dependent. Here, we sought to identify amino acids involved in picrotoxin inhibition of 5-HT3 receptors. Mutation of serine to alanine at the transmembrane domain 2 (TM2) 2' position did not affect picrotoxin (PTX) sensitivity in murine 5-HT3A receptors. However, mutation of the 6' TM2 threonine to phenylalanine dramatically reduced PTX sensitivity. Mutation of 6' asparagine to threonine in the 5-HT3B subunit enhanced PTX sensitivity in heteromeric 5-HT3A/3B receptors. Introduction of serine (native to the human 3B subunit) at the 6' position also increased PTX sensitivity, suggesting a species-specific effect. Mutation of the 7' leucine to threonine in 5-HT3A receptors increased PTX sensitivity roughly 10-fold, comparable with that observed in GABA(A) receptors, and also conferred distinct gating kinetics. The equivalent mutation in the 3B subunit (i.e., 7' valine to threonine) had no impact on PTX sensitivity in 5-HT3A/3B receptors. Interestingly, [3H]ethynylbicycloorthobenzoate ([3H]EBOB), a high-affinity ligand to the convulsant site in GABA(A) receptors, did not exhibit specific binding in 5-HT3A receptors. The structurally related compound, tert-butylbicyclophosphorothionate (TBPS), which potently inhibits GABA(A) receptors, did not inhibit 5-HT3 currents. Our results indicate that the TM2 6' residue is a common determinant of PTX inhibition of both 5-HT3 and GABA(A) receptors and demonstrate a role of the 7' residue in PTX inhibition. However, lack of effects of EBOB and TBPS in 5-HT3A receptors suggests that the functional domains in the two receptors are not equivalent and underscores the complexity of PTX modulation of LGICs.

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.

We have previously shown that extracellular protons inhibit recombinant and native GABA(A) receptors. In this report, we studied the site(s) and mechanism by which protons modulate the GABA(A) receptor. Whole cell GABA-activated currents were recorded from human embryonic kidney (HEK) 293 cells expressing recombinant alpha1beta2gamma2 GABA(A) receptors. Protons competitively inhibited the response to GABA and bicuculline. In contrast, change in pH did not influence direct gating of the channel by pentobarbital, and it did not influence spontaneous channel openings in alpha1(L264T)beta2gamma2 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABA(A) receptors at the ligand binding site, we systemically mutated N-terminal residues known to be involved in GABA binding and assessed effects of pH on these mutant receptors. Site-specific mutation of beta2 Y205 to F or alpha1 F64 to A, both of which are known to influence GABA binding, significantly reduced pH sensitivity of the GABA response. These mutations did not affect Zn(2+) sensitivity, suggesting that H(+) and Zn(2+) do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethylpyrocarbonate (DEPC) reduced Zn(2+)-mediated inhibition of GABA(A) receptors but had no effect on proton-induced inhibition of GABA currents. In addition, mutation of residues known to be involved in Zn(2+) modulation had no effect on pH modulation of GABA(A) receptors. Our results support the hypothesis that protons inhibit GABA(A) receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H(+) and Zn(2+) in GABA(A) receptors are distinct.

Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA(A) receptors. Lactones with small alkyl substitutions at the alpha-position positively modulate the channel, whereas beta-substituted lactones tend to inhibit the GABA(A) receptor. These compounds mediate inhibition through the picrotoxin site of the receptor. A distinct binding site that mediates the stimulatory actions of lactones is presumed to exist, although no definitive evidence to support this claim exists. In the present study, we used in vivo and in vitro assays to evaluate the effects of the enantiomers of a novel lactone, alpha-benzyl-alpha-methyl-gamma-butyrolactone (alpha-BnMeGBL), on the GABA(A) receptor. R-(-)-alpha-BnMeGBL was 2-fold more potent than the S-(+)-alpha-BnMeGBL in blocking pentylenetetrazol-induced seizures in CF-1 mice. The (+)-enantiomer inhibited binding of t-butylbicyclophosporothionate with a higher affinity than the (-)-enantiomer (IC(50) of 0.68 and 1.1 mM, respectively). Whole cell patch-clamp recordings from recombinant alpha1beta2gamma2 receptors stably expressed in HEK293 cells demonstrated that both compounds stimulated GABA-activated current. The maximal stimulation was approximately 2-fold greater with (+)-alpha-BnMeGBL than that seen with (-)-alpha-BnMeGBL. Both enantiomers of alpha-BnMeGBL directly gated the GABA(A) receptor at mM concentrations, in a nonstereoselective manner. Our data demonstrate the stimulatory actions of alpha-BnMeGBL on GABA(A) receptor function display enantioselectivity and provide strong evidence for the existence of a true "lactone site" on the receptor.

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.

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. Nitrendipine's 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.

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

There are currently no known agents that display selectivity between homomeric 5-hydroxytryptamine type 3A (5-HT3A) and heteromeric 5-HT3A/3B receptors. In the present study, we show that the CNS convulsant picrotoxin selectively interacts with 5-HT3A receptors. In whole-cell patch clamp recordings, the inhibitory effect of PTX was reduced 100-fold in heteromeric mouse 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in both native tissue and recombinant receptor preparations.

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA(A) receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA(A) receptors composed of either alpha1beta2gamma2Lor alpha1beta2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor beta2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor beta2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA(A) receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.

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.

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABA(A) receptors are formed from a number of distinct subunits, designated alpha, beta, gamma, delta, epsilon, and theta, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABA(A) receptors present on hypothalamic neurons. To gain insight into which GABA(A) receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABA(A) receptor-mediated currents were detected in all neurons tested and had an average EC(50) of 20 +/- 1.6 microM. Hypothalamic GABA(A) receptors were modulated by diazepam (EC(50) = 0.060 microM), zolpidem (EC(50) = 0.19 microM), loreclezole (EC(50) = 4.4 microM), methyl-6,7-dimethoxy-4-ethyl-beta-carboline (EC(50) = 7.7 microM), and 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). Conversely, these receptors were inhibited by Zn(2+) (IC(50) = 70.5 microM), dehydroepiandrosterone sulfate (IC(50) = 16.7 microM), and picrotoxin (IC(50) = 2.6 microM). The alpha4/6-selective antagonist furosemide (10-1,000 microM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABA(A) receptor subtypes that incorporate alpha1 and/or alpha2 subunits, beta2 and/or beta3 subunits, and the gamma2 subunit. Our results suggest receptors expressing alpha3-alpha6, beta1, gamma1, and delta, if present, represent a minor component of functional hypothalamic GABA(A) receptors.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA(A) and glutamate-gated Cl(minus sign) channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA(C) receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine alpha1 receptors from non-use-facilitated to use-facilitated. In wild type alpha1 receptors, PTX inhibited glycine-gated Cl(minus sign) current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15'-serine to glutamine (alpha1(S15'Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in alpha1(S15'Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in alpha1(S15'N) receptors. The reciprocal mutation in the beta2 subunit of alpha1beta2 GABA(A) receptors (alpha1beta2(N15'S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels.

To gain insight into specific GABA(A) receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1-14 day old rats using whole-cell patch clamp recordings. GABA(A-) receptor-mediated currents were detected in all neurons tested, with an average EC(50) of 22.2 microM. GABA currents were consistently stimulated by diazepam (EC(50)=63 nM), zolpidem (EC(50)=85 nM), loreclezole (EC(50)=10.1 microM) and the neurosteroid 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn(2+) (IC(50)=33.6 microM) picrotoxin (IC(50)=2.4 microM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10-1000 microM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant alpha subunit isoform present in GABA(A) receptors of the NTS appears to be the alpha1 and/or alpha2 subunit. beta2 and/or beta3 subunits are the major beta isoform, while the predominant gamma subunit is likely gamma2. Our data suggest the contribution to NTS GABA currents by alpha3-alpha6, beta1, gamma1 and delta subunits, if present, is minor by comparison.

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the gamma-aminobutyric acid type A (GABA(A)) receptor. In the present study, we have investigated the mechanism and site of action of PTZ in recombinant GABA(A) receptors. In rat alpha 1 beta 2 gamma 2 receptors, PTZ inhibited GABA-activated Cl(-) current in a concentration-dependent, voltage-independent manner, with an IC(50) of 0.62 +/- 0.13 mM. The mechanism of inhibition appeared competitive with respect to GABA in both rat and human alpha 1 beta 2 gamma 2 receptors. Varying subunit configuration (change or lack of alpha subunit isoform or lack of gamma 2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC(50) values (0.6-2.2 mM) in all receptor configurations tested. This contrasts with PTX and other PTX-site ligands, which have greater affinity in receptors lacking an alpha subunit. Using a one-site model for PTZ interaction with alpha 1 beta 2 gamma 2 receptors, the association rate (k(+1)) was found to be 1.14 x 10(3) M(-1) s(-1) and the dissociation rate (k(-1)) was 0.476 s(-1), producing a functional k(d) of 0.418 mM. PTZ could only gain access to its binding site extracellularly. Single-channel recordings demonstrated that PTZ decreased open probability by increasing the duration of closed states but had no effect on single-channel conductance or open state duration. alpha-Isopropyl-alpha-methyl-gamma-butyrolactone, a compound known to antagonize effects of PTX, also diminished the effects of PTZ. Taken together, our results indicate that pentylenetetrazole and picrotoxin interact with overlapping but distinct domains of the GABA(A) receptor.

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.

We tested the ability of the central nervous system convulsant pentylenetetrazole (PTZ) to inhibit gamma-aminobutyric acid (GABA)-gated current in receptors expressing a mutation that rendered them resistant to picrotoxin. Consistent with previous reports, receptors expressing beta2(T246F), along with alpha3 and gamma2 subunits, resulted in a greatly diminished sensitivity to picrotoxin. Sensitivity to PTZ was completely abolished in the mutant receptor, confirming the hypothesis that PTZ acts at the picrotoxin site. Quite unexpected, however, was our finding that PTZ elicited marked stimulation (up to 400% of control) in the mutated receptors. This stimulatory effect was not mediated via an interaction with the benzodiazepine site, as preincubation with the benzodiazepine antagonist flumazenil did not block the PTZ-induced stimulation. Our results reveal the existence of a novel stimulatory domain of PTZ in GABA(A) 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 picrotoxin's 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.

We studied the effects of extracellular pH (pHo) on gamma-aminobutyric acid (GABA)-mediated Cl- current in rat hypothalamic neurons and recombinant type-A GABA (GABA(A)) receptors stably expressed in human embryonic kidney cells (HEK 293), using whole cell and outside-out patch-clamp recordings. In alpha3beta2gamma2s receptors, acidic pH decreased, whereas alkaline pH increased the response to GABA in a reversible and concentration-dependent manner. Acidification shifted the GABA concentration-response curve to the right, significantly increasing the EC50 for GABA without appreciably changing the slope or maximal current induced by GABA. We obtained similar effects of pH in alpha1beta2gamma2 receptors and in GABA-activated currents recorded from thin hypothalamic brain slices. In outside-out patches recorded from alpha3beta2gamma2 recombinant receptors, membrane patches were exposed to 5 microM GABA at control (7.3), acidic (6.4), or alkaline (8.4) pH. GABA activated main and subconductance states of 24 and 16 pS, respectively, in alpha3beta2gamma2 receptors. Alkaline pH(o) increased channel opening frequency and decreased the duration of the long closed state, resulting in an increase in open probability (from 0.0801 +/- 0.015 in pH 7.3 to 0.138 +/- 0.02 in pH 8.4). Exposure of the channels to acidic pH(o) had the opposite effects on open probability (decreased to 0.006 +/- 0.0001). Taken together, our results indicate that the function of GABA(A) receptors is modulated by extracellular pH. The proton effect is similar in recombinant and native receptors and is dependent on GABA concentration. In addition, the effect appears to be independent of the alpha-subunit isoform, and is due to the ability of H+ to alter the frequency of channel opening. Our findings indicate that GABAergic signaling in the CNS may be significantly altered during conditions that increase or decrease pH.

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., (Copyright 1999 Elsevier Science B.V.)

Previous reports from this laboratory have shown that a high percentage of neurons in the caudal hypothalamus are stimulated by hypoxia both in vivo and in vitro. This stimulation is in the form of an increase in firing frequency and significant membrane depolarization. The goal of the present study was to determine if this hypoxia-induced excitation is influenced by development. In addition, we sought to determine the mechanism by which hypoxia stimulates caudal hypothalamic neurons. Caudal hypothalamic neurons from neonatal (4-16 days) or juvenile (20-40 days) rats were patch-clamped, and the whole cell voltage and current responses to moderate (10% O2) or severe (0% O2) hypoxia were recorded in the brain slice preparation. Analysis of tissue oxygen levels demonstrated no significant difference in the levels of tissue oxygen in brain slices between the different age groups. A significantly larger input resistance, time constant and half-time to spike height was observed for neonatal neurons compared with juvenile neurons. Both moderate and severe hypoxia elicited a net inward current in a significantly larger percentage of caudal hypothalamic neurons from rats aged 20-40 days (juvenile) as compared with rats aged 4-16 days (neonatal). In contrast, there was no difference in the magnitude of the inward current response to moderate or severe hypoxia between the two age groups. Those cells that were stimulated by hypoxia demonstrated a significant decrease in input resistance during hypoxic stimulation that was not observed in those cells unaffected by hypoxia. A subset of neurons were tested independent of age for the ability to maintain the inward current response to hypoxia during synaptic blockade (11.4 mM Mg2+/0. 2 mM Ca2+). Most of the neurons tested (88.9%) maintained a hypoxic excitation during synaptic blockade, and this inward current response was unaffected by addition of 2 mM cobalt chloride to the bathing medium. In contrast, perfusion with the Na+ channel blocker, tetrodotoxin (1-2 microM) or Na+ replacement with N-methyl-D-glucamine (NMDG) significantly reduced the inward current response to hypoxia. Furthermore, the input resistance decrease observed during hypoxia was attenuated significantly during perfusion with NMDG. These results indicate the excitation elicited by hypoxia in hypothalamic neurons is age dependent. In addition, the inward current response of caudal hypothalamic neurons is not dependent on synaptic input but results from a sodium-dependent conductance.

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.

Despite the presence of the multiple subunits (alpha, beta, gamma, and delta) and their isoforms for gamma-aminobutyric acid, type A (GABAA) receptors in mammalian brains, the alpha x beta 2 gamma 2 subtypes appear to be the prototype GABAA receptors sharing many properties with native neuronal receptors. In order to gain insight into their subunit stoichiometry and orientation, we prepared a tandem construct of the alpha 6 and beta 2 subunit cDNAs where the carboxyl-terminal of alpha 6 is linked to the amino-terminal of beta 2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cells with the tandem construct alone failed to induce GABA-dependent CI- currents, but its cotransfection with the cDNA for alpha 6 or gamma 2, but not beta 2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of gamma 2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [3H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the beta 2. No appreciable differences were found in their Kd (2.5 nM) and Bmax values (1.4 pmol/mg of protein). These data are consistent with the view that the polypeptides arising from the tandem construct were expressed with the high affinity GABA site, but unable to form GABA channels. The requirement of a specific monomeric subunit (alpha 6 or gamma 2) for the tandem construct to express Cl--currents supports a pentameric structure of GABAA receptors consisting of two alpha 6, two beta 2, and one gamma 2 for the alpha 6 beta 2 gamma 2 and three alpha 6 and two beta 2 for the alpha 6 beta 2 subtype.

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.

Various GABAA receptor subunits share four highly homologous putative transmembrane domains (M1 to M4) and have been proposed to form an ion channel of pentameric structure with M2 lining the pore. The carboxyl terminal side of M2 contains three amino acid residues containing a hydroxyl group, which are Thr 265, 266 and serine 268 in the alpha 6 subunit. In order to study their functional role, we generated mutants of the alpha 6 subunit carrying a single point mutation of threonine 265 or 266 to alanine, or serine 268 to glycine. Co-expression of the mutants with beta 2 and gamma 2 subunits in human embryonic kidney cells produced functional receptors which are similar to the wild type in their sensitivity to a benzodiazepine agonist (U-92330), insensitivity to Zn, anion permeability, and GABA dose-response profiles as monitored with the whole cell patch clamp technique. Only in the alpha 6T266A beta 2 gamma 2 subtype, however, GABA-induced Cl- currents decayed much more rapidly than the wild type (about 10 times faster). Analysis of the GABA dependency of desensitization indicates that the T266A mutation enhanced the desensitization rate with little effect on the recovery rate from desensitization or on the half-maximal GABA concentration. We conclude that threonine 266 in the alpha 6 subunit plays a pivotal role in desensitization processes of GABAA receptors.

U-93631,[4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline], represents a GABAA receptor ligand of novel chemical structure, and has been shown to induce a rapid, time-dependent decay of GABA-induced whole-cell Cl- currents in recombinant GABAA receptors (Dillon et al., 1993). In this study, we found that the drug competitively inhibited [35S]t-butylbicyclophosphorothionate binding to the picrotoxin site on a cloned GABAA receptor, alpha 1 beta 2 gamma 2, and preempted the action of picrotoxin and [35S]t-butylbicyclophosphorothionate on GABA-induced Cl- currents. We further examined the effect of U-93631 on GABA-induced single channel openings in outside-out patches. U-93631 (5 microM) showed no effect on single channel conductance, the duration of channel open states or the short closed state, but increased the duration of the long closed state and its relative contribution to total closed time. In addition, closings of very long duration (> 500 msec), albeit rare, occurred more frequently in the presence of U-93631. Thus, the probability of single channel openings decreased from 0.12 +/- 0.02 to 0.04 +/- 0.01 in the presence of U-93631 (5 microM). These properties of U-93631 are analogous to those of picrotoxin and [35S]t-butylbicyclophosphorothionate reported in earlier studies with native neurons. We conclude that U-93631 at least shares overlapping binding domains with picrotoxin and [35S]t-butylbicyclophosphorothionate, and the ability to stabilize the GABAA receptor/Cl- channel complex in an inactivated state(s).(ABSTRACT TRUNCATED AT 250 WORDS)

We discovered the ability of U-93631 (4-dimethyl-3-t-butylcarboxyl-4,5- dihydro[1,5-a]imidazoquinoxaline) to accelerate decay of gamma-aminobutyric acid (GABA)-induced currents, and we explored its mechanism in human embryonic kidney cells (HEK-293) stably expressing the alpha 1 beta 2 gamma 2 subtype of GABAA receptors. Inward currents (Cl- efflux) induced by 5 microM GABA at the holding potential of -60 mV (under a symmetrical Cl- gradient) decayed with an exponential time course with a mean time constant (tau) of 222 +/- 25 sec, as examined with the whole-cell configuration of the patch-clamp technique. The monoexponential decay was greatly accelerated in the presence of U-93631 at 5 microM, with the mean tau value being 5.2 +/- 0.5 sec. The tau values were dependent on the concentration of U-93631, with an estimated Kd of approximately 2 microM. Outward currents at the holding potential of +60 mV decayed with a similar tau value in the presence of the drug, suggesting the voltage independence of the drug action. The initial amplitude of the GABA (5 microM)-induced Cl- current was not affected by preincubation with U-93631 (5 microM) or GABA (200 nM) alone but was reduced by preincubation with the combination of the two. In the presence of U-93631 at 5 microM, the peak amplitude decreased as a function of GABA concentration, with the half-maximal inhibitory concentration being approximately 100 nm, which is close to the Kd for the high affinity GABA site (85 nM). It appears that the drug interacts with GABA-bound receptors (at least monoliganded) and accelerates receptor desensitization, rather than acting as an open channel blocker. The binding site for U-93631 on GABAA receptors seems not to overlap with GABA, barbiturate, or benzodiazepine sites, because the drug effect persisted in the presence of excess ligands for those sites. With cloned GABAA receptors composed of only alpha 1 beta 2, beta 2 gamma 2, or alpha 1 gamma 2 subunits, U-93631 also accelerated the decay rate. This lack of subtype selectivity raises the possibility that the compound interacts with a region common among the three subunits, probably a novel modulatory site, which can possibly be exploited as a novel therapeutic target.

Several studies have suggested that the caudal hypothalamus modulates responses to hypercapnia and hypoxia. In addition, this area of the hypothalamus contains neurons that have a sympathoexcitatory discharge. The purpose of the present study was to determine whether the basal discharge of caudal hypothalamic neurons that are stimulated by hypercapnia or hypoxia is related to cardiovascular (sympathetic discharge and/or the cardiac cycle) and/or respiratory activity (phrenic nerve discharge). Hypothalamic single unit activity, phrenic nerve activity, and cervical sympathetic nerve activity were recorded in anesthetized cats. Computer averaging techniques were used to compare temporally the discharge of hypothalamic neurons with cardiovascular and/or respiratory activity. Cardiorespiratory and hypothalamic neuronal responses to ventilation with hypoxic (10% O2/90% N2) and hypercapnic (5% CO2/95% O2) gases were determined in intact and in peripherally-chemodenervated, barodenervated cats. Thirty-two percent of hypothalamic neurons were stimulated by a hypercapnic stimulus in intact cats; of those that were stimulated by hypercapnia, all had a basal discharge related to cardiovascular and/or respiratory activity. Hypoxia significantly increased the discharge rate of 21% of hypothalamic units in intact animals; 90% of those had a cardiovascular and/or respiratory-related rhythm. Only 13% of the neurons were stimulated by both hypoxia and hypercapnia. Similar results were found in barodenervated, peripherally chemodenervated cats. Neurons excited by these stimuli in both the intact and denervated cats were found to be concentrated in the posterior hypothalamic area. The results of this study suggest that a group of caudal hypothalamic neurons contribute to the cardiorespiratory responses to hypoxia and hypercapnia, but via separate subpopulations of neurons. In addition, input from peripheral baroreceptor and chemoreceptor afferents is not required for this modulation.