Single-channel Open Time Research Articles

Distinct Functional Alterations and Therapeutic Options of Two Pathological De Novo Variants of the T292 Residue of GABRA1 Identified in Children with Epileptic Encephalopathy and Neurodevelopmental Disorders.

Mutations of GABAAR have reportedly led to epileptic encephalopathy and neurodevelopmental disorders. We have identified a novel de novo T292S missense variant of GABRA1 from a pediatric patient with grievous global developmental delay but without obvious epileptic activity. This mutation coincidentally occurs at the same residue as that of a previously reported GABRA1 variant T292I identified from a pediatric patient with severe epilepsy. The distinct phenotypes of these two patients prompted us to compare the impacts of the two mutants on the receptor function and to search for suitable therapeutics. In this study, we used biochemical techniques and patch-clamp recordings in HEK293 cells overexpressing either wild-type or mutated rat recombinant GABAARs. We found that the α1T292S variant significantly increased GABA-evoked whole-cell currents, shifting the dose–response curve to the left without altering the maximal response. In contrast, the α1T292I variant significantly reduced GABA-evoked currents, shifting the dose–response curve to the right with a severely diminished maximum response. Single-channel recordings further revealed that the α1T292S variant increased, while the α1T292I variant decreased the GABAAR single-channel open time and open probability. Importantly, we found that the T292S mutation-induced increase in GABAAR function could be fully normalized by the negative GABAAR modulator thiocolchicoside, whereas the T292I mutation-induced impairment of GABAAR function was largely rescued with a combination of the GABAAR positive modulators diazepam and verapamil. Our study demonstrated that α1T292 is a critical residue for controlling GABAAR channel gating, and mutations at this residue may produce opposite impacts on the function of the receptors. Thus, the present work highlights the importance of functionally characterizing each individual GABAAR mutation for ensuring precision medicine.

Recruiting RyRs to Open in a Ca2+ Release Unit: Single-RyR Gating Properties Make RyR Group Dynamics

In cardiac myocytes, clusters of type-2 ryanodine receptors (RyR2s) release Ca2+ from the sarcoplasmic reticulum (SR) via a positive feedback mechanism in which fluxed Ca2+ activates nearby RyRs. Although the general principles of this are understood, less is known about how single-RyR gating properties define the RyR group dynamics in an array of many channels. Here, we examine this using simulations with three models of RyR gating that have identical open probabilities: the commonly used two-state Markov gating model, one that utilizes multiple exponentials to fit single-channel open time (OT) and closed time (CT) distributions, and an extension of this multiexponential model that also includes experimentally measured correlations between single-channel OTs and CTs. The simulations of RyR clusters that utilize the multiexponential gating model produce infrequent Ca2+ release events with relatively few open RyRs. Ca2+ release events become even smaller when OT/CT correlations are included. This occurs because the correlations produce a small but consistent bias against recruiting more RyRs to open during the middle of a Ca2+ release event, between the initiation and termination phases (which are unaltered compared to the uncorrelated simulations). In comparison, the two-state model produces frequent, large, and long Ca2+ release events because it had a recruitment bias in favor of opening more RyRs. This difference stems from the two-state model’s single-RyR OT and CT distributions being qualitatively different from the experimental ones. Thus, the details of single-RyR gating can profoundly affect SR Ca2+ release even if open probability and mean OTs and CTs are identical. We also show that Ca2+ release events can terminate spontaneously without any reduction in SR [Ca2+], luminal regulation, Ca2+-dependent inactivation, or physical coupling between RyRs when Ca2+ flux is below a threshold value. This supports and extends the pernicious attrition/induction decay hypothesis that SR Ca2+ release events terminate below a threshold Ca2+ flux.

Pathophysiology of and therapeutic options for a GABRA1 variant linked to epileptic encephalopathy

We report the identification of a de novo GABRA1 (R214C) variant in a child with epileptic encephalopathy (EE), describe its functional characterization and pathophysiology, and evaluate its potential therapeutic options. The GABRA1 (R214C) variant was identified using whole exome sequencing, and the pathogenic effect of this mutation was investigated by comparing wild-type (WT) α1 and R214C α1 GABAA receptor-expressing HEK cells. GABA-evoked currents in these cells were recorded using whole-cell, outside-out macro-patch and cell-attached single-channel patch-clamp recordings. Changes to surface and total protein expression levels of WT α1 and R214C α1 were quantified using surface biotinylation assay and western blotting, respectively. Finally, potential therapeutic options were explored by determining the effects of modulators, including diazepam, insulin, and verapamil, on channel gating and receptor trafficking of WT and R214C GABAA receptors. We found that the GABRA1 (R214C) variant decreased whole-cell GABA-evoked currents by reducing single channel open time and both surface and total GABAA receptor expression levels. The GABA-evoked currents in R214C GABAA receptors could only be partially restored with benzodiazepine (diazepam) and insulin. However, verapamil treatment for 24 h fully restored the function of R214C mutant receptors, primarily by increasing channel open time. We conclude that the GABRA1 (R214C) variant reduces channel activity and surface expression of mutant receptors, thereby contributing to the pathogenesis of genetic EE. The functional restoration by verapamil suggests that it is a potentially new therapeutic option for patients with the R214C variant and highlights the value of precision medicine in the treatment of genetic EEs.

Type I-like behavior of the type II α7 nicotinic acetylcholine receptor positive allosteric modulator A-867744.

Cognitive impairment often involves the decreased expression or hypofunction of alpha 7-type nicotinic acetylcholine receptors (α7 nAChRs). Agonists or positive allosteric modulators (PAMs) of α7 nAChRs are known to be potential treatments for dementias, different neurodegenerative disorders, pain syndromes and conditions involving inflammation. In some of these conditions, it is desirable to maintain the temporal precision of fast cholinergic events, while in others, this temporal precision is unnecessary. For this reason, the optimal therapeutic effect for distinct indications may require PAMs with different mechanisms of action. The two major mechanisms are called “type I”, which are compounds that augment α7 nAChR-mediated currents but maintain their characteristic fast kinetics; and “type II”, which are compounds that produce augmented and prolonged currents. In this study, we performed a kinetic analysis of two type II PAMs of the α7 nAChR: PNU-120596 and A-867744, using a fast perfusion method that allowed high temporal resolution. We characterized the type of modulation produced by the two compounds, the state-dependence of the modulatory action, and the interaction between the two compounds. We found fundamental differences between the modulation mechanisms by PNU-120596 and A-867744. Most importantly, during brief agonist pulses, A-867744 caused a strikingly type I-like modulation, while PNU-120596 caused a type II-like prolonged activation. Our results demonstrate that specific compounds, even though all labeled as type II PAMs, can behave in completely different ways, including their onset and offset kinetics, state preference, and single channel open time. Our results emphasize that subtle details of the mechanism of action may be significant in assessing the therapeutic applicability of α7 nAChR PAM compounds.

Triheteromeric GluN1/GluN2A/GluN2C NMDARs with Unique Single-Channel Properties Are the Dominant Receptor Population in Cerebellar Granule Cells

NMDA-type glutamate receptors (NMDARs) are ligand-gated ion channels that mediate excitatory neurotransmission in the CNS. Here we describe functional and single-channel properties of triheteromeric GluN1/GluN2A/GluN2C receptors, which contain two GluN1, one GluN2A, and one GluN2C subunits. This NMDAR has three conductance levels and opens in bursts similar to GluN1/GluN2A receptors but with a single-channel open time and open probability reminiscent of GluN1/GluN2C receptors. The deactivation time course of GluN1/GluN2A/GluN2C receptors is intermediate to GluN1/GluN2A and GluN1/GluN2C receptors and is not dominated by GluN2A or GluN2C. We show that triheteromeric GluN1/GluN2A/GluN2C receptors are the predominant NMDARs in cerebellar granule cells and propose that co-expression of GluN2A and GluN2C in cerebellar granule cells occludes cell surface expression of diheteromeric GluN1/GluN2C receptors. This new insight into neuronal GluN1/GluN2A/GluN2C receptors highlights the complexity of NMDAR signaling in the CNS.

P.016 A novel de novo GABRA1 mutation linked to epileptic encephalopathy: pathophysiology and potential therapeutic options

Background: Epileptic encephalopathy (EE) is a severe neurological disorder characterized by treatment-resistant seizures and poor neurodevelopmental outcomes. EE is associated with mutant genes, including those that encode for γ-aminobutyric acid type A (GABAA) receptor subunits. We identified a novel de novo GABRA1 mutation in a patient with EE, characterized its impact on GABAA receptor function, and sought potential therapeutic options. Methods: We described the clinical and electrophysiological features of a patient with a novel de novo GABRA1 (R214C) mutation; performed functional studies; and determined the effect of diazepam and insulin on wild type and mutant GABAA receptors. Results: The patient is a 10-year-old girl with EE, treatment-resistant seizures, intellectual disability and autism. Her GABRA1 (R214C) mutation dramatically decreased whole-cell GABA-evoked currents by reducing GABAA surface receptors, decreasing single channel open time, and altering channel kinetic properties. The combination of diazepam and insulin partially repaired these effects by enhancing channel activity and increasing the number of surface receptors, respectively. Conclusions: Diazepam and insulin partially mitigated a de novo GABRA1 (R214C) mutation’s effects on GABAA receptor number and function. Given the risks of insulin use, pharmacological agents with similar mechanisms of action but fewer side effects, such as IGF-1, should be studied and considered for clinical application.

Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

Apomorphine Is a Bimodal Modulator of TRPA1 Channels

Apomorphine is a non-narcotic derivative of morphine, which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from Parkinson's disease. Adverse effects of apomorphine treatment include severe emesis and nausea, and ulceration and pain at the injection site. We wanted to test whether sensory transient receptor potential (TRP) channels are a molecular target for apomorphine. Here, we show that rTRPV1, rTRPV2, rTRPV3, and mTRPV4, as well as hTRPM8, and rTRPM3, which are expressed in dorsal root ganglion neurons, are insensitive toward apomorphine treatment. This also applied to the cellular redox sensor hTRPM2. On the contrary, human TRPA1 could be concentration-dependently modulated by apomorphine. Whereas the addition of apomorphine in the low micromolar range produced an irreversible activation of the channel, application of higher concentrations caused a reversible voltage-dependent inhibition of heterologously expressed TRPA1 channels, resulting from a reduction of single-channel open times. In addition, we provide evidence that apomorphine also acts on endogenous TRPA1 in cultured dorsal root ganglion neurons from rats and in the enterochromaffin model cell line QGP-1, from which serotonin is released upon activation of TRPA1. Our study shows that human TRPA1 is a target for apomorphine, suggesting that an activation of TRPA1 might contribute to adverse side effects such as nausea and painful injections, which can occur during treatment with apomorphine.

Activation of α7 Nicotinic Receptors by Orthosteric and Allosteric Agonists: Influence on Single-Channel Kinetics and Conductance

Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (~160-800-fold, respectively), and shut times are shorter (~8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ~2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations.

Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.

Conserved Structural and Functional Control of N-Methyl-d-aspartate Receptor Gating by Transmembrane Domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.

Regulation of single NMDA receptor channel activity by alpha-actinin and calmodulin in rat hippocampal granule cells.

The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability (P(open)), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in P(open). The physiological implications of these findings are discussed.

Modulation of single channels underlying hippocampal L-type current enhancement by agonists depends on the permeant ion.

The influx of calcium (Ca(2+)) ions through L-type channels underlies many cellular processes, ranging from initiation of gene transcription to activation of Ca(2+)-activated potassium channels. L-type channels possess a diagnostic pharmacology, being enhanced by the dihydropyridine BAY K 8644 and benzoylpyrrole FPL 64176. It is assumed that the action of these compounds is independent of the ion conducted through the channel. In contrast to this assumption, modulation of L-type channel activity in acutely dissociated rat CA1 hippocampal neurons depended on the divalent ion identity. BAY K 8644 and FPL 64176 substantially increased single-channel open time only when barium (Ba(2+)) was the permeant ion. BAY K 8644 increased single-channel conductance when either Ba(2+) or Ca(2+) ions were the charge carrier, an effect not observed with FPL 64176. BAY K 8644 enhanced the whole cell L-type channel Ca(2+)- or Ba(2+)-carried current without a change in deactivation tail kinetics. In contrast, enhancement by FPL 64176 was associated with a dramatic slowing of deactivation kinetics only when Ba(2+) and not Ca(2+) was the charge carrier. Current activation was slowed by FPL 64176 with either charge carrier, an effect arising from a clustering of agonist-modified long-duration openings toward the end of the voltage step. These data indicate that agonists enhanced L-type current by distinct mechanisms dependent on the permeant ion, indicating that care must be considered when used as diagnostic tools.

An arginine involved in GABA binding and unbinding but not gating of the GABA(A) receptor.

GABA(A) receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose-response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function. Here, we focused on a residue (beta2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of beta2-R207, we expressed alpha1beta2 and alpha1beta2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because beta2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Mechanisms underlying modulation of neuronal KCNQ2/KCNQ3 potassium channels by extracellular protons.

Changes in extracellular pH occur during both physiological neuronal activity and pathological conditions such as epilepsy and stroke. Such pH changes are known to exert profound effects on neuronal activity and survival. Heteromeric KCNQ2/3 potassium channels constitute a potential target for modulation by H+ ions as they are expressed widely within the CNS and have been proposed to underlie the M-current, an important determinant of excitability in neuronal cells. Whole-cell and single-channel recordings demonstrated a modulation of heterologously expressed KCNQ2/3 channels by extracellular H+ ions. KCNQ2/3 current was inhibited by H+ ions with an IC50 of 52 nM (pH 7.3) at −60 mV, rising to 2 μM (pH 5.7) at −10 mV. Neuronal M-current exhibited a similar sensitivity. Extracellular H+ ions affected two distinct properties of KCNQ2/3 current: the maximum current attainable upon depolarization (Imax) and the voltage dependence of steady-state activation. Reduction of Imax was antagonized by extracellular K+ ions and affected by mutations within the outer-pore turret, indicating an outer-pore based process. This reduction of Imax was shown to be due primarily to a decrease in the maximum open-probability of single KCNQ2/3 channels. Single-channel open times were shortened by acidosis (pH 5.9), while closed times were increased. Acidosis also recruited a longer-lasting closed state, and caused a switch of single-channel activity from the full-conductance state (∼8 pS) to a subconductance state (∼5 pS). A depolarizing shift in the activation curve of macroscopic KCNQ2/3 currents and single KCNQ2/3 channels was caused by acidosis, while alkalosis caused a hyperpolarizing shift. Activation and deactivation kinetics were slowed by acidosis, indicating specific effects of H+ ions on elements involved in gating. Contrasting modulation of homomeric KCNQ2 and KCNQ3 currents revealed that high sensitivity to H+ ions was conferred by the KCNQ3 subunit.

Mutations linked to generalized epilepsy in humans reduce GABA A receptor current

The first mutations of the GABA A receptor channel linked to familial epilepsy in humans were reported in the γ2L subunit (Baulac et al., 2001; Wallace et al., 2001). When expressed in Xenopus oocytes, the γ2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and the γ2 subunit K289M mutation decreased current amplitudes. In this study, single channel recording and concentration jump techniques were used to evaluate the effects of these mutations on GABA A receptor currents from receptors expressed in human embryonic kidney cells (Bianchi et al., 2002). When co-expressed with α1 and β3 subunits, the mutated γ2 subunits did not alter current activation rates or rates or extent of desensitization during prolonged (400 ms) GABA application (1 mM). Deactivation following brief (5 ms) or prolonged (400 ms) GABA application was accelerated for the K289M, but not the R43Q mutation. Single channel analysis showed shorter mean open duration, suggesting that the faster deactivation was likely caused by altered gating efficacy. Interestingly, the R43Q mutation did not alter diazepam potentiation. However, significantly smaller current amplitudes were observed that were not explained by decreased single channel conductance or open time, suggesting this mutation resulted in reduced surface expression of functional receptors. These two γ mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of normal neuronal inhibition depends on both the peak amplitude of IPSCs and their time course.

Functional maturation of nicotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system.

Under normal conditions in situ, muscle fibers and motoneurons, the main partners of motor units, are strongly dependent on each other. This interdependence hinders ex vivo studies of neuromuscular disorders where nervous or muscular components are considered separately. To allow in vitro access to complex nerve-muscle relationships, we developed a novel nerve-muscle co-culture system where mouse muscle innervation is assured by rat spinal cord explants. The degree of muscular maturation during co-culture was evaluated using the distribution of nicotinic acetylcholine receptors (AChRs) and their electrophysiological characteristics before and after innervation. In myotubes from non-innervated cultures, AChRs were diffusely distributed over the entire myotube surface. Their single-channel conductance (33.5+/-0.6 pS) and mean open time (8.1+/-0.7 ms) are characteristic of AChRs described in embryonic or denervated skeletal muscles. In innervated muscle fibers from co-cultures, AChRs appear as discrete aggregates and co-localize with synaptotagmin. In addition to the embryonic type currents, in innervated fibers AChR currents having high conductance (53.3+/-5.9 pS) and short mean open time (2.6+/-0.1 ms), characteristic of AChRs at mature neuromuscular junctions, were observed. Our data support the use of this new nerve-muscle co-culture system as a reliable model for the study of murine muscular differentiation and function.

Kinetic and pharmacological properties of GABA(A) receptors in single thalamic neurons and GABA(A) subunit expression.

Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic gamma-aminobutyric acid type A (GABA(A)) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei-the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABA(A) channels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 +/- 2.5 vs. 3.8 +/- 0.7 (SE) ms, respectively]. GABA(A) receptors in nRt and VB cells were pharmacologically distinct. Zn(2+) (100 microM) reduced GABA(A) channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed alpha1-alpha3, alpha5, beta1-3, gamma2-3, and delta, while nRt cells expressed alpha3, alpha5, gamma2-3, and delta. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABA(A) receptor heterogeneity within individual thalamic neurons. beta subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABA(A) receptors on beta subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding beta subunits, our findings do support alpha subunits, possibly alpha1 versus alpha3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the gamma2 and delta subunits previously implicated in Zn(2+) sensitivity are both expressed in each cell type, the observed differential Zn(2+) actions at VB versus nRt GABA(A) receptors may involve other subunit differences.

Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors.

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.

The effects of pH on the interaction between capsaicin and the vanilloid receptor in rat dorsal root ganglia neurons.

1. The vanilloid receptor of sensory neurons is a polymodal nociceptor sensitive to capsaicin, protons, heat and anandamide. Although it is known that interaction occurs between these different mediators the mechanism by which this occurs is poorly understood. In this study capsaicin elicited currents were recorded from vanilloid receptors found in adult rat isolated dorsal root ganglia (DRG) neurons under conditions of varying pH and the mechanism whereby protons can modulate this capsaicin response investigated. 2. Under whole-cell voltage clamp, modulating extracellular pH shifted the position of the capsaicin log(concentration)-response curve. Acidification from pH 9.0 to pH 5.5 lowered the EC50 values from 1150+/-250 nM to 5+/-2 nM with coincident change in the mean apparent slope factor from 2.3+/-0.3 to 0.9+/-0.2 and no change in maximal response. 3. The magnitude of the potentiation seen on reducing extracellular pH was not significantly affected by changes in extracellular calcium and magnesium concentration. 4. The response to capsaicin was not potentiated by a reduction in intracellular pH suggesting a site of action more accessible from the extracellular than the intracellular side of the membrane. 5. Potentiation by low pH was voltage independent indicating a site of action outside the membrane electric field. 6. At the single channel level, reducing extracellular pH increased channel open probability but had no significant effect on single channel conductance or open time. 7. These results are consistent with a model in which, on reducing extracellular pH, the vanilloid receptor in rat DRG neurons, changes from a state with low affinity for capsaicin to one with high affinity, coincident with a loss of cooperativity. This effect, presumed to be proton mediated, appears to involve one or more sites with pK(a) value 7.4-7.9, outside the membrane electrical field on an extracellularly exposed region of the receptor protein.