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The [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] and [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] two isoforms of [gamma]-aminobutyric acid type A ([GABA.sub.A]) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] channels exhibited briefer active periods than [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels was ~2.6 [micro]M compared with ~19 [micro]M for [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] channels, suggesting that GABA binds to the [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of [GABA.sub.A] receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate [GABA.sub.A] receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system. doi/ 10.1085/jgp.200910317

Millimolar concentrations of the barbiturate pentobarbital (PB) activate [gamma]-aminobutyric acid (GABA) type A receptors (GABARs) and cause blockade reported by a paradoxical current increase or 'tail' upon washout. To explore the mechanism of blockade, we investigated PB-triggered currents of recombinant [[alpha].sub.1][[beta].sub.2] [[gamma].sub.2S] GABARs in whole cells and outside-out membrane patches using rapid perfusion. Whole cell currents showed characteristic bell-shaped concentration dependence where high concentrations triggered tail currents with peak amplitudes similar to those during PB application. Tail current time courses could not be described by multi-exponential functions at high concentrations ([greater than or equal to] 3,000 [micro]M). Deactivation time course decayed over seconds and was slowed by increasing PB concentration and application time. In contrast, macropatch tail currents manifested eightfold greater relative amplitude, were described by multi-exponential functions, and had millisecond rise times; deactivation occurred over fractions of seconds and was insensitive to PB concentration and application time. A parsimonious gating model was constructed that accounts for macropatch results ('patch' model). Lipophilic drug molecules migrate slowly through cells due to avid partitioning into lipophilic subcellular compartments. Inclusion of such a pharmacokinetic compartment into the patch model introduced a slow kinetic component in the extracellular exchange time course, thereby providing recapitulation of divergent whole cell results. GABA co-application potentiated PB blockade. Overall, the results indicate that block is produced by PB concentrations sixfold lower than for activation involving at least three inhibitory PB binding sites, suggest a role of blocked channels in GABA-triggered activity at therapeutic PB concentrations, and raise an important technical question regarding the effective rate of exchange during rapid perfusion of whole cells with PB.

The family of [gamma]-aminobutyric acid type A receptors ([GABA.sub.A]Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic [GABA.sub.A]Rs that are mainly comprised of [[alpha].sub.1], [[beta].sub.2], and [[gamma].sub.2] subunits, whereas tonic inhibition is mediated by extrasynaptic [GABA.sub.A]Rs comprised of [[alpha].sub.4/6], [[beta].sub.2], and [delta] subunits. We investigated the activation properties of recombinant [[alpha].sub.4][[beta].sub.2] [delta] and [[alpha].sub.1][[beta].sub.2][gamma].sub.2s] [GABA.sub.A]Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3 (2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at [[alpha].sub.4][[beta].sub.2][delta] [GABA.sub.A]Rs ([beta] ~ 1600 [s.sup.-1]) than at [[alpha].sub.1][[beta].sub.2] [gamma].sub.2s] [GABA.sub.A]Rs ([beta] ~ 460 [s.sup.-1]), whereas GABA activated [[alpha].sub.1][[beta].sub.2][gamma].sub.2s] [GABA.sub.A]Rs more rapidly ([beta] ~1800 [s.sup.-1]) than [[alpha].sub.4][[beta].sub.2][delta] [GABA.sub.A]Rs ([beta] < 440 [s.sup.-1). Single channel recordings of [[alpha].sub.1] [[beta].sub.2][gamma].sub.2s] and [[alpha].sub.4][[beta].sub.2] [delta] [GABA.sub.A]Rs showed that both channels open to a main conductance state of ~25 pS at -70 mV when activated by GABA and low concentrations of THIE whereas saturating concentrations of THIP elicited ~36 pS openings at both channels. Saturating concentrations of GABA elicited brief (

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the [alpha]-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-[HT.sub.3A]) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-[HT.sub.3A]-glvM3M4 receptors were comparable to 5-[HT.sub.3A] wild type. The channels remained cation-selective but the 5-[HT.sub.3A]-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-[HT.sub.3] and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-[HT.sub.3A] but not 5-[HT.sub.3A]-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-[rho]1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.

Selective permeability is a fundamental property of ion channels. The Cys-loop receptor superfamily is composed of both excitatory (ACh, 5-HT) and inhibitory (GABA, glycine) neurotransmitter-operated ion channels. In the GABA receptor, it has been previously shown that the charge selectivity of the integral pore can be altered by a single mutation near the intracellular end of the second transmembrane-spanning domain (TM2). We have extended these findings and now show that charge selectivity of the anionic [rho]1 GABA receptor can be influenced by the introduction of glutamates, one at a time, over an 8-amino acid stretch (-2' to 5') in the proposed intracellular end of TM2 and the TM1-TM2 intracellular linker. Depending on the position, glutamate substitutions in this region produced sodium to chloride permeability ratios ([P.sub.Na]+/[sub.Ck]-) varying from 0.64 to 3.4 (wild type [P.sub.Na] + [sub./Cl]- = 0). In addition to providing insight into the mechanism of ion selectivity, this functional evidence supports a model proposed for the homologous nicotinic acetylcholine receptor in which regions of the protein, in addition to TM2, form the ion pathway.

The influence of extracellular calcium ions and other divalent metal ions on the activity of the gamma amino butyric acid receptor c (GABAc) of the horizontal cells of the catfish retina was evaluated using patch clamp analysis. Results indicate that calcium has an excitatory effect while zinc has an inhibitory effect on GABAc responses. The implications of these findings on the regulation of events in the horizontal cells are discussed.

The primary structure of the membrane-spanning segment of the gamma-aminobutyric receptor was characterized by mutating group residues within the subunit. The study showed that nine amino acid residues project into the channel opening and may participate in binding reactions. The primary structure also points to a helical secondary structure for the subunit that ends in a threonine residue.