Open-channel Blocking Mechanism Research Articles

Kinetic mechanism of NMDA receptor inhibition by the ketamine metabolite 2R,6R-hydroxynorketamine.

2R,6R-hydroxynorketamine (2R,6R-HNK) is a metabolite of the fast-acting dissociative anesthetic ketamine (KET). It is currently in clinical trials for the treatment of major depressive disorder due to its strong safety profile at larger doses. The endogenous therapeutic target of 2R,6R-HNK is unknown. We reported recently that like the parent compound ketamine, 2R,6R-HNK reduces NMDA receptor currents with an open-channel block mechanism which is voltage-dependent, pH-dependent, and competitive with Mg2+, and has ∼100-fold lower efficacy (IC50, 46 µM) than KET (IC50, 0.5 µM) at pH 7.2 and −70 mV. We used electrophysiology, kinetic analyses, and kinetic modeling to examine the effect of 2R,6R-HNK on the reaction mechanism of NMDA receptors. We recorded on-cell single-channel currents from HEK293 cells expressing GluN1/GluN2A receptors (and GFP) with pipettes containing (in mM): 150 NaCl, 2.5 KCl, 1 EDTA, 10 HEPES, pH 7.2 (NaOH), agonists glutamate (1) and glycine (0.1), and applied +100 mV (membrane potential ∼−120 mV). Relative to activity recorded in the absence of 2R,6R-HNK (control, n=7), activity recorded with 50 µM 2R,6R-HNK (n=7) displayed a lower open probability (Po), shorter openings (MOT), and longer closed durations (MCT). Fitting these data with state models allowed us to optimize rate constants for the kinetic model. In the future, we will test these models by simulating macroscopic responses and recording whole cell currents in similar conditions.

Mechanism of open-channel block in the chloride channel TMEM16A

TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis, but its pharmacology remains poorly understood. Here, we present a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and a detailed functional analysis of its inhibition mechanism. A defined binding pocket located immediately external to the neck region of the hourglass-shaped pore is responsible for open-channel block by 1PBC and presumably chemically similar compounds.

Molecular mechanisms of action determine inhibition of paroxysmal depolarizing shifts by NMDA receptor antagonists in rat cortical neurons

N-methyl-d-aspartate glutamate receptors (NMDARs) are involved in numerous central nervous system (CNS) processes, including epileptiform activity. We used a picrotoxin-induced epileptiform activity model to compare the action of different types of NMDAR antagonists in rat brain slices. Paroxysmal depolarizing shifts (PDS) were evoked by external stimulation in the medial prefrontal cortex (mPFC) slices and recorded in pyramidal cells (PC) and in fast-spiking interneurons (FSI). The NMDAR antagonists APV and memantine reduced the duration of PDS. However, the competitive antagonist APV caused similar effects on the PC and FSI, while the open-channel blocker memantine had a much stronger effect on the PDS in the FSI than in the PC. This difference cannot be explained by a corresponding difference in NMDAR sensitivity to memantine because the drug inhibited the excitatory postsynaptic current (EPSC) similarly in both cell types. Importantly, the PDS were significantly longer in the FSI than in the PC. The degree of PDS inhibition by memantine correlated with individual PDS durations in each cell type. Computer modeling of a synaptic network in the mPFC suggests that the different effects of memantine on the PDS in the PC and FSI can be explained by use dependence of its action. An open-channel blocking mechanism and competition with Mg2+ ions for the binding site result in pronounced inhibition of the long PDS, whereas the short PDS are weakly sensitive. Our results show that peculiarities of kinetics and the mechanism of action largely determine the effects of NMDAR antagonists on physiological and/or pathological processes.

Cocaine potently blocks neuronal α3β4 nicotinic acetylcholine receptors in SH-SY5Y cells.

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3β4-nAChR expressed in native SH-SY5Y cells. α3β4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 μM. Kinetic analysis showed that cocaine accelerated α3β4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 μM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-βS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 μM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3β2β3-nAChRs and α4β2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3β4-nAChRs expressed in SH-SY5Y cells.

Inhibition of the NMDA and AMPA receptor channels by antidepressants and antipsychotics

It is known that some antidepressants and antipsychotics directly inhibit NMDA-type ionotropic glutamate receptors. In this study we systematically studied action of seven drugs (Fluoxetine, Citalopram, Desipramine, Amitriptyline, Atomoxetine, Chlorpromazine, and Clozapine) on NMDA receptors and Ca2+-permeable and -impermeable AMPA receptors in rat brain neurons by whole-cell patch-clamp technique. Except for weak effect of fluoxetine, all drugs were virtually inactive against Ca2+-impermeable AMPA receptors. Fluoxetine and desipramine significantly inhibited Ca2+-permeable AMPA receptors (IC50=43±7 and 105±12µM, respectively). Desipramine, atomoxetine and chlorpromazine inhibited NMDA receptors in clinically relevant low micromolar concentrations, while citalopram had only weak effect. All tested medicines have been clustered into two groups by their action on NMDA receptors: desipramine, amitriptyline, chlorpromazine, and atomoxetine display voltage- and magnesium-dependent open channel blocking mechanism. Action of fluoxetine and clozapine was found to be voltage- and magnesium-independent. All voltage-dependent compounds could be trapped in closed NMDA receptor channels. Possible contribution of NMDA receptor inhibition by certain antidepressants and antipsychotics to their analgesic effects in neuropathic pain is discussed.

Pharmacologically targeted NMDA receptor antagonism by NitroMemantine for cerebrovascular disease.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in ‘Big Pharma,’ ‘undruggable’ for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders.

Open channel block of NMDA receptors by diphenhydramine

Background and purposeDiphenhydramine is a well known H1-receptor antagonist that plays a major role in clinical practice. Nowadays, diphenhydramine is primarily applied to prevent nausea but also its sedative and analgesic effects are of clinical importance. As other drugs mediating sedative and analgesic properties partly operate via the inhibition of glutamate receptors, we tested the hypothesis that diphenhydramine, as well interacts with excitatory ionotropic glutamate receptors. Experimental approachElectrophysiological patch-clamp experiments were performed on glutamate receptors which were heterologously expressed in human TsA cells. Key resultsDiphenhydramine inhibits NMDA-mediated membrane currents in a reversible and concentration-dependent manner at clinically relevant concentrations. The inhibition occurred in a noncompetitive manner. Diphenhydramine did not compete with NMDA or glycine for their binding sites and half-maximal inhibition was obtained around 25 μM diphenhydramine, independent of the subunit composition. The inhibition was caused by a classical open channel blocking mechanism and varied strongly with the membrane potential. Our results suggest that diphenhydramine most probably interacts with the Mg2+ binding site or a very closely related area of the channel pore. Conclusion and implicationsThe data presented here provide evidence that the NMDA receptor antagonism of diphenhydramine contribute to its sedative and potentially LTP-related effects like analgesia and amnesia.

Dissecting the Inhibitory and Potentiation Effects of Desformylflustrabromine on a Muscle-Type Nicotinic Acetylcholine Receptor (NACHR)

Desformylflustrabromine (dFBr) potentiates 4β2 nAChR in vitro (Kim et al. 2007, Biorg. Med. Chem. Lett. 17: 4855) and reduces nicotine self-administration in vivo (Liu 2013; Psychopharmacology 230: 203). We have reported that dFBr is a potent inhibitor (IC50, ∼1 M) of human (Hα12β1eδ) and Torpedo (Tα12β1γδ) muscle-type nAChRs. We also found, using [3H]dFBr reversible binding and dFBr displacement of [3H]PCP binding, that dFBr binds with high affinity to the muscle-type nAChRs ion channel in the desensitized state. But, when we photolabeled nAChR-rich Torpedo membranes with [3H]dFBr in the presence of agonist, four dFBr binding sites were evident. [3H]dFBr photolabeled amino acids in the ion channel and in three binding sites within the extracellular domain previously identified for nAChRs PAMs galanthamine and physostigmine. The high affinity binding of dFBr in the ion channel precludes the ability to study the functional contribution of dFBr binding within the extracellular domain and to determine whether dFBr acts as inhibitor or potentiator at these sites. Therefore, we are introducing mutations in the muscle-type nAChR ion channel M2 helices to eliminate the channel blocking effect of dFBr. The effect of M2 mutations on the EC50 of ACh and on dFBr inhibition of muscle-type nAChR were examined using two-electrode voltage clamp recording. Serine substitutions of the leucine at M2-9 of each nAChR subunit decreased dFBr IC50 compared with wild-type, consistent with an open channel blocking mechanism. This results confirm our [3H]dFBr the photolabeling results demonstrating that dFBr binds within the ion channel in close proximity to M2-9. The effects of other substitutions at M2-9 and at other positions in the M2 helices on dFBr inhibition of muscle-type nAChR are being tested.

Diminazene Interaction with ASIC1A Channels

Diaryldiamidines, a class of DNA minor groove binders used clinically since the 1940s in the treatment of tropical parasitoses such as trypanosomiasis, leishmaniasis, pneumocystis pneumonia and babesiosis, have been recently found to exert specific blocking effects on acid-sensing ion channels (ASIC), raising hopes to develop new small molecule neuroprotective agents for ischemic stroke, which involves acidosis-mediated activation of ASIC1a and heteromeric ASIC1a/2b channels. Therefore we have thoroughly investigated the modes of interaction of diminazene with ASIC1a via whole-cell and single-channel recordings on outside-out patches excised from HEK293 cells of ASIC1a transient currents evoked by brief acidic pulses (pH 6.0). Data idealization and model-dependent fitting were performed with custom hidden Markov model maximum likelihood algorithms. We proved that the 3 subconductance levels are due to partial block by calcium of the permeation pathway, and expanded our previous 8-state linear connectivity gating model (Marin et al. 2008 Channels 2(6):1-10) including 3 subconductance states for each of the 2 gating modes. Single-channel recordings with diminazene 0.6 or 3 μM indicated a mechanism of open channel block. By exponential fits of the distributions of blocked and unblocked periods for each gating mode we computed binding and unbinding rates and dissociation constants of 0.13 and 0.27 μM for gating modes 2 and 1, respectively. Using atomic resolution models of human ASIC1a in the open conformations corresponding to the 2 gating modes, we found via molecular docking several intra- and intersubunit binding sites for diminazene. One of these sites, assumed to be responsible for the state-dependent block, features protrusion of the drug in the transmembrane domain permeation pathway. As expected, for this blocking site the interaction energy is higher in the symmetrical open conformation of the second gating mode than in the asymmetrical open conformation of the first gating mode.

Mechanism of Estradiol-Induced Block of Voltage-Gated K+ Currents in Rat Medial Preoptic Neurons

The present study was conducted to characterize possible rapid effects of 17-β-estradiol on voltage-gated K+ channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-β-estradiol affects the K+ channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-β-estradiol rapidly (within seconds) and reversibly reduced the K+ currents, showing an EC50 value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca2+, and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K+ currents, membrane-impermeant forms of estradiol did not reduce the K+ currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the KV-2-channel blocker r-stromatoxin-1. The time course of K+ current in 17-β-estradiol, with a time-dependent inhibition and a slight dependence on external K+, suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-β-estradiol binds to open K+ channels. It was concluded that 17-β-estradiol rapidly reduces voltage-gated K+ currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.

A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

Resurgent currents turn painfully exciting

Resurgent currents turn painfully exciting

Effects of lobeline, a nicotinic receptor ligand, on the cloned Kv1.5

The goal of the present study was to examine the effects of lobeline, an agonist at nicotinic receptors, on cloned Kv channels, Kv1.5, Kv3.1, Kv4.3, and human ether-a-gogo-related gene (HERG), which are stably expressed in Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. Whole-cell patch-clamp experiments revealed that lobeline accelerated the decay rate of Kv1.5 inactivation, decreasing the current amplitude at the end of the pulse in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) value of 15.1 μM. Using a time constant for the time course of drug-channel interaction, the apparent association (k(+1)), and dissociation rate (k(-1)) constants were 2.4 μΜ(-1) s(-1) and 40.9 s(-1), respectively. The calculated K(D) was 17.0 μΜ. Lobeline slowed the decay rate of the tail current, resulting in a tail crossover phenomenon. The inhibition of Kv1.5 by lobeline steeply increased at potentials between -10 and +10 mV, which corresponds to the voltage range of channel activation. At more depolarized potentials a weaker voltage dependence was observed (δ=0.26). The voltage dependence of the steady-state activation curve was not affected by lobeline, but lobeline shifted the steady-state inactivation curve of Kv1.5 in the hyperpolarizing direction. Lobeline produced use-dependent inhibition of Kv1.5 at frequencies of 1 and 2 Hz, and slowed the recovery from inactivation. Lobeline also inhibited Kv3.1, Kv4.3, and HERG in a concentration-dependent manner, with IC(50) values of 21.7, 28.2, and 0.34 μM, respectively. These results indicate that lobeline produces a concentration-, time-, voltage-, and use-dependent inhibition of Kv1.5, which can be interpreted as an open-channel block mechanism.

The Anticonvulsive Drug Lamotrigine Blocks Neuronal α4β2 Nicotinic Acetylcholine Receptors

Lamotrigine (LTG), an anticonvulsive drug, is often used for the treatment of a variety of epilepsies. In addition to block of sodium channels, LTG may act on other targets to exert its antiepileptic effect. In the present study, we evaluated the effects of LTG on neuronal nicotinic acetylcholine receptors (nAChRs) using the patch-clamp technique on human α4β2-nAChRs heterologously expressed in the SH-EP1 cell line and on native α4β2-nAChRs in dopaminergic (DA) neurons in rat ventral tegmental area (VTA). In SH-EP1 cells, LTG diminished the peak and steady-state components of the inward α4β2-nAChR-mediated currents. This effect exhibited concentration-, voltage- and use-dependent behavior. Nicotine dose-response curves showed that in the presence of LTG, the nicotine-induced maximal current was reduced, suggesting a noncompetitive inhibition. These findings suggest that LTG inhibits human neuronal α4β2-nAChR function through an open-channel blocking mechanism. LTG-induced inhibition in α4β2-nAChRs was more profound when preceded by a 2-min pretreatment, after which the nicotine-induced current was reduced even without coapplication of LTG, suggesting that LTG is also able to inhibit α4β2-nAChRs without channel activation. In freshly dissociated VTA DA neurons, LTG inhibited α4β2-nAChR-mediated currents but did not affect glutamate- or GABA-induced currents, indicating that LTG selectively inhibits nAChR function. Collectively, our data suggest that the neuronal α4β2-nAChR is likely an important target for mediating the anticonvulsive effect of LTG and the blockade of α4β2-nAChR possibly underlying the mechanism through which LTG effectively controls some types of epilepsy, such as autosomal dominant nocturnal frontal lobe epilepsy or juvenile myoclonic epilepsy.

Block and allosteric modulation of GABAergic currents by oenanthotoxin in rat cultured hippocampal neurons

Oenanthotoxin (OETX), a polyacetylenic alcohol from plants of the genus Oenanthe, has recently been identified as potent inhibitor of GABA-evoked currents. However, the effects of OETX on the inhibitory postsynaptic currents (IPSCs), as well as the pharmacological mechanism(s) underlying its effects on GABA(A) receptors, remain unknown. The purpose of this study was to elucidate the mechanism underlying the inhibition of GABAergic currents by OETX. Effects of OETX on GABAergic currents were studied using the patch clamp technique on rat cultured hippocampal neurons. Miniature IPSCs (mIPSCs) were recorded in the whole-cell configuration, while the current responses were elicited by ultrafast GABA applications onto the excised patches. OETX potently inhibited both mIPSCs and current responses, but its effect was much stronger on synaptic currents. Analysis of the effects of OETX on mIPSCs and evoked currents disclosed a complex mechanism: allosteric modulation of both GABA(A) receptor binding and gating properties and a non-competitive, probably open channel block mechanism. In particular, OETX reduced the binding rate and nearly abolished receptor desensitization. A combination of rapid clearance of synaptic GABA and OETX-induced slowing of binding kinetics is proposed to underlie the potent action of OETX on mIPSCs. OETX shows a complex blocking mechanism of GABA(A) receptors, and the impact of this toxin is more potent on mIPSCs than on currents evoked by exogenous GABA. Such effects on GABAergic currents are compatible with the convulsions and epileptic-like activity reported for OETX.

Atomoxetine acts as an NMDA receptor blocker in clinically relevant concentrations

There is increasing evidence that not only the monoaminergic but also the glutamatergic system is involved in the pathophysiology of attention-deficit hyperactivity disorder (ADHD). Hyperactivity of glutamate metabolism might be causally related to a hypoactive state in the dopaminergic system. Atomoxetine, a selective noradrenaline reuptake inhibitor, is the first non-stimulant approved for the treatment of this disorder. Here we have evaluated the effects of atomoxetine on glutamate receptors in vitro. The whole-cell configuration of the patch-clamp technique was used to analyse the effect of atomoxetine on N-methyl-d-aspartate (NMDA) receptors in cultured rodent cortical and hippocampal neurons as well as on NMDA receptors heterologously expressed in human TsA cells. Atomoxetine blocked NMDA-induced membrane currents. Half-maximal inhibition emerged at about 3 microM which is in the range of clinically relevant concentrations found in plasma of patients treated with this drug. The inhibition was voltage-dependent, indicating an open-channel blocking mechanism. Furthermore, the inhibitory potency of atomoxetine did not vary when measured on NMDA receptors from different brain regions or with different subunit compositions. The effective NMDA receptor antagonism by atomoxetine at low micromolar concentrations may be relevant to its clinical effects in the treatment of ADHD. Our data provide further evidence that altered glutamatergic transmission might play a role in ADHD pathophysiology.

The Interaction of the Neuroprotective Compounds Riluzole and Phenobarbital with AMPA-Type Glutamate Receptors: A Patch-Clamp Study

Background: Blockade of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors is a promising pharmacological strategy in the treatment of neurodegenerative diseases. The aim of the study is to elucidate if there are direct interactions of riluzole and phenobarbital with AMPA-type receptor channels and to determinethe molecular pharmacological mechanisms. Methods: The patch-clamp technique was used combining an ultrafast solution exchange system to investigate the interaction of riluzole and phenobarbital with recombinant AMPA-type glutamate receptor channels (homomeric GluR2flipGQ or nondesensitizing GluR2L504Y). Results: A dose-dependent decrease in the relative peak current amplitude (rAmp) and the relative area-under-the-current curve (rAUC) were found after preincubation with 0.1 mmol/l or higher concentrations of riluzole. Furthermore, in coapplication experiments with GluR2L504Y, the application of 1 or 3 mmol/l riluzole showed a decrease in the current decay time constant, and a reopening current was observed at 3 mmol/l riluzole. Phenobarbital blocks AMPA receptor channels dose-dependently in the coapplication experiments, and reopening currents after removing glutamate and blocker were observed. A slight block effect after preincubation should indicate an additional competitive block effect. Conclusion: Riluzole and phenobarbital modulate AMPA-type receptor channels separately, which could be both characterized as a combination of open-channel block and competitive-block mechanism.

Emerging Roles ofS-Nitrosylation in Protein Misfolding and Neurodegenerative Diseases

Overactivation of N-methyl-D-aspartate (NMDA)-type glutamate receptors accounts, at least in part, for excitotoxic neuronal damage, potentially contributing to a wide range of acute and chronic neurologic disorders. Recent studies have suggested that generation of excessive nitric oxide (NO) and reactive oxygen species (ROS) can mediate excitotoxicity, in part by triggering protein misfolding. S-Nitrosylation, which is a covalent reaction of a NO group with a cysteine thiol, represents one such mechanism that can contribute to NO-induced neurotoxicity. The ubiquitin-proteasome system (UPS), in conjunction with molecular chaperones, can prevent accumulation of aberrantly-folded proteins. For example, protein disulfide isomerase (PDI) can provide neuroprotection from misfolded proteins or endoplasmic reticulum stress through its molecular chaperone and thiol-disulfide oxidoreductase activities. Here, the authors present recent evidence suggesting that NO contributes to degenerative conditions by S-nitrosylating PDI (forming SNO-PDI) and the ubiquitin protein ligase, parkin (forming SNO-parkin). Moreover, it is demonstrated for the first time that inhibition of excessive NMDA receptor activity by memantine, via a mechanism of uncompetitive open-channel block with a relatively rapid off-rate, can ameliorate excessive production of NO, protein misfolding, and neurodegeneration.

Valerenic acid potentiates and inhibits GABA A receptors: Molecular mechanism and subunit specificity

Valerian is a commonly used herbal medicinal product for the treatment of anxiety and insomnia. Here we report the stimulation of chloride currents through GABA A receptors (I GABA) by valerenic acid (VA), a constituent of Valerian. To analyse the molecular basis of VA action, we expressed GABA A receptors with 13 different subunit compositions in Xenopus oocytes and measured I GABA using the two-microelectrode voltage-clamp technique. We report a subtype-dependent stimulation of I GABA by VA. Only channels incorporating β 2 or β 3 subunits were stimulated by VA. Replacing β 2/3 by β 1 drastically reduced the sensitivity of the resulting GABA A channels. The stimulatory effect of VA on α 1β 2 receptors was substantially reduced by the point mutation β 2N265S (known to inhibit loreclezole action). Mutating the corresponding residue of β 1 (β 1S290N) induced VA sensitivity in α 1β 1S290N comparable to α 1β 2 receptors. Modulation of I GABA was not significantly dependent on incorporation of α 1, α 2, α 3 or α 5 subunits. VA displayed a significantly lower efficiency on channels incorporating α 4 subunits. I GABA modulation by VA was not γ subunit dependent and not inhibited by flumazenil (1 μM). VA shifted the GABA concentration–effect curve towards lower GABA concentrations and elicited substantial currents through GABA A channels at ≥30 μM. At higher concentrations (≥100 μM), VA and acetoxy-VA inhibit I GABA. A possible open channel block mechanism is discussed. In summary, VA was identified as a subunit specific allosteric modulator of GABA A receptors that is likely to interact with the loreclezole binding pocket.

Open Channel Block by Ca2+ Underlies the Voltage Dependence of Drosophila TRPL Channel

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca2+ blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca2+ block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca2+ that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca2+ affinity are active.