Martin Wallner

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABAA receptor (GABAR), α6β3δ, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in α6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of α6β3δ GABARs. Behavioral and electrophysiological comparisons of Gabra6100R/100R and Gabra6100Q/100Q rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.

A calcium switch for the functional coupling between α (hslo) and β subunits (KV, Caβ) of maxi K channels

K V , Ca β subunit dramatically increases the apparent calcium sensitivity of the α subunit of MaxlK channels when probed in the micromolar [Ca2+]i range. Analysis in a wide range of [Ca2+]i revealed that this functional coupling is exquisitely modulated by [Ca2+]i. Ca2+ ions switch MaxiK α+β complex into a functionally coupled state at concentrations beyond resting [Ca2+]i. At [Ca2+] ≤ 100 nM, MaxiK activity becomes independent of Ca2+, is purely voltage‐activated, and its functional coupling with its β subunit is released. The functional switch develops at [Ca2+]i that occur during cellular excitation, providing the molecular basis of how MaxiK channels regulate smooth muscle excitability and neurotransmitter release.

Ethanol enhances α4β3δ and α6β3δ γ-aminobutyric acid type A receptors at low concentrations known to affect humans

γ-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (≥60 mM). We show that GABARs containing the δ-subunit, which are highly sensitive to γ-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR δ-subunit, which is thought to be associated exclusively with α4- and α6-subunits in vivo, and the β3-subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing β2-instead of β3-subunits in α4βδ- and α6βδ-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing γ2-instead of δ-subunits with α4β and α6β are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that “extrasynaptic” δ-subunit-containing GABARs, but not their “synaptic” γ-subunit-containing counterparts, are primary targets for EtOH.

Ethanol enhances alpha 4 beta 3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low concentrations known to affect humans.

γ-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (≥60 mM). We show that GABARs containing the δ-subunit, which are highly sensitive to γ-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR δ-subunit, which is thought to be associated exclusively with α4- and α6-subunits in vivo, and the β3-subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing β2-instead of β3-subunits in α4βδ- and α6βδ-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing γ2-instead of δ-subunits with α4β and α6β are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that “extrasynaptic” δ-subunit-containing GABARs, but not their “synaptic” γ-subunit-containing counterparts, are primary targets for EtOH.

Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors

Extrasynaptic GABA(A) receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits-either α4 or α6-two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC(50) of 30-50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP.

Contributions of the GABAA Receptor α6 Subunit to Phasic and Tonic Inhibition Revealed by a Naturally Occurring Polymorphism in the α6 Gene

GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit α6 (Gabra6100R and Gabra6100Q). Depending on their subunit partners, these two variants of α6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6100R/100R and Gabra6100Q/100Q rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by δ subunit-containing GABARs in Gabra6100Q/100Q rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates α6/δ subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is ∼160 nm in perfused slices. These results (1) demonstrate contributions of α6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that δ subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

Physiology and pharmacology of alcohol: the imidazobenzodiazepine alcohol antagonist site on subtypes of GABAA receptors as an opportunity for drug development?

Alcohol (ethanol, EtOH) has pleiotropic actions and induces a number of acute and long‐term effects due to direct actions on alcohol targets, and effects of alcohol metabolites and metabolism. Many detrimental health consequences are due to EtOH metabolism and metabolites, in particular acetaldehyde, whose high reactivity leads to nonspecific chemical modifications of proteins and nucleic acids. Like acetaldehyde, alcohol has been widely considered a nonspecific drug, despite rather persuasive evidence implicating inhibitory GABAA receptors (GABAARs) in acute alcohol actions, for example, a GABAAR ligand, the imidazobenzodiazepine Ro15‐4513 antagonizes many low‐to‐moderate dose alcohol actions in mammals. It was therefore rather surprising that abundant types of synaptic GABAARs are generally not responsive to relevant low concentrations of EtOH. In contrast, δ‐subunit‐containing GABAARs and extrasynaptic tonic GABA currents mediated by these receptors are sensitive to alcohol concentrations that are reached in blood and tissues during low‐to‐moderate alcohol consumption. We recently showed that low‐dose alcohol enhancement on highly alcohol‐sensitive GABAAR subtypes is antagonized by Ro15‐4513 in an apparently competitive manner, providing a molecular explanation for behavioural Ro15‐4513 alcohol antagonism. The identification of a Ro15‐4513/EtOH binding site on unique GABAAR subtypes opens the possibility to characterize this alcohol site(s) and screen for compounds that modulate the function of EtOH/Ro15‐4513‐sensitive GABAARs. The utility of such drugs might range from novel alcohol antagonists that might be useful in the emergency room, to drugs for the treatment of alcoholism, as well as alcohol‐mimetic drugs to harness acute positive effects of alcohol.

Modulation of the skeletal muscle sodium channel α-subunit by the β1 -subunit

Co‐expression of cloned sodium channel β1 ‐subunit with the rat skeletal muscle‐subunit (αμI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from , indicating that β1 restores ‘physiological’ behaviour. Northern blot analysis revealed that the Na+ channel β1‐subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the ‘brain’ β1 cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na + channel α‐subunits in brain, skeletal muscle and heart may share a common β1 ‐subunit.

Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

GABAA receptors (GABAARs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABAARs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn2+ block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABAARs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABAARs.

Chapter 8 Calcium-Activated Potassium Channels in Muscle and Brain

Publisher Summary This chapter discusses the characterization of the two subfamilies that can account for the variety of Ca 2+- sensitive K + channels: large conductance (BK) and small conductance (SK) channels. BK channels are characterized by their high single-channel conductance, their dual response to voltage and Ca 2+ , and their blockade by nanomolar concentrations of iberiotoxin and micromolar concentrations of TEA. Heterologously expressed α-subunit cDNA clones mirror these features. Structurally, they are characterized by unique N- and C-terminal sequences, implicated in β-subunit and Ca 2+ regulation, appended to a six-transmembrane structure typical of voltage-gated ion channels. SK channels are characterized by their small conductance, their voltage independence, and their Ca 2+ -dependent activation. A family of this class of K + channels has been recently cloned. BK and different types of SK channels are often coexpressed within the same native cell and seem to be coupled to specific Ca 2+ sources. Specific Ca 2+ channel blockers can abolish certain types of calcium-activated K channels without affecting others.