High conductance ␥-aminobutyric acid type A (GABA A ) channels (>40 picosiemens (pS)) have been reported in some studies on GABA A channels in situ but not in others, whereas recombinant GABA A channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA A channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA A channels. GABARAP, a protein associated with native GABA A channels, has been reported to cause clustering of GABA A receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing ␣ 1 , ␤ 1 , and ␥ 2S subunits of human GABA A receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA A receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.

The single channel properties of recombinant ␥-aminobutyric acid type A (GABA A ) ␣␤␥ receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing ␣␤␥ receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA A receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (>40 pS), and GABA (>10 M) was able to increase their conductance, up to a maximum of 60 pS Inhibitory signals in human brains are mediated primarily by ␥-aminobutyric acid type A (GABA A ) 2 receptors. These ligandgated ion channels are composed of multimembrane-spanning subunits that assemble into pentamers and function by gating a pore selective for chloride ions. The targeting and organization of GABA A receptors at specific membrane locations are critical for their normal function. For example, GABA A receptors are clustered at inhibitory synapses but are also found both clustered and nonclustered at other sites on the neuronal cell surface (1, 2). These synaptic and nonsynaptic (extrasynaptic) sites reflect GABA A receptor involvement in both phasic and tonic signaling, respectively. The functional behavior of native GABA A receptors is complex. Much of the receptor's functional complexity has been attributed to its extensive structural heterogeneity as indicated by the 19 different genes identified to date (␣1-6, ␤1-3, ␥1-3, ␦, 1-3, ⑀, , and ).Recombinant GABA A receptors are different from native GABA A receptors in that they never display single channel conductances greater than 40 pS, nor do drugs modulate their conductance, properties we and others have described for native nonsynaptic (extrasynaptic) GABA A receptors (3-8). We have, however, been able to mimic the behavior exhibited by neuronal extrasynaptic GABA A receptors in a recombinant system and change the dispersion of receptors in the membrane simply by co-expressing the trafficking protein GABARAP with GABA A receptors (9). GABARAP (GABA A receptor-associated protein) was originally identified because of its physical association with GABA A receptors following their isolation by immunoprecipitation from solubilized rat brain (10). Subsequent immunolocalization data and the biochemical identification of the GABARAP interaction partners has led to the suggestion that it participates in trafficking and membrane fusion events underlying organizational processes at GABAergic synapses but does not remain associated with receptors once they are inserted at the synapse (11). In heterologous expression systems, recombinant GABARAP has been shown to promote clustering of ␥ subunit-containing GABA A receptors in the plasma membrane (9, 12). As a consequence of this ordered packing arrangement, the recombinant GABA ...

GABA(A) receptors composed of alpha, beta and gamma subunits display a significantly higher single-channel conductance than receptors comprised of only alpha and beta subunits. The pore of GABA(A) receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6', 10' and 13' positions. At the 2' position, however, a polar residue is present in the gamma subunit but not the alpha or beta subunits. As residues at the 2', 6' and 10' positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type alpha1beta1 and alpha1beta1gamma2S receptors with two mutant receptors, alphabetagamma(S2'A) and alphabetagamma(S2'V). We found that the single-channel conductance of both mutant alphabetagamma receptors was significantly decreased with respect to wild-type alphabetagamma, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant alphabetagamma receptors remained larger than wild-type alphabeta channels. This reduction in the conductance of mutant alphabetagamma receptors was observed at depolarized potentials only (E(Cl) = -1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the gamma2' residue. The substitutions at the gamma2' serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2' position in M2 of the gamma subunit affects both single-channel conductance and receptor kinetics.

Ligand-gated ion channels respond to specific neurotransmitters by transiently opening an integral membrane ion-selective pore, allowing ions to move down their electrochemical gradient. A distinguishing feature of all members of the ligand-gated ion channel superfamily is the presence of a 13-amino acid disulfide loop (Cys-loop) in the extracellular ligand-binding domain. Structural data derived from the acetylcholine receptor place this loop at the interface between the ligand-binding domain and the transmembrane pore-forming domain where it is ideally located to participate in coupling ligand binding to channel opening. We have introduced specific mutations into a conserved motif at the mid-point of the Cys-loop of the GABA A receptor subunits alpha1, beta2 and gamma2S where the sequence reads aromatic, proline, aliphatic (ArProAl motif). Receptors carrying a mutation in the Cys-loop of one of their subunits were expressed in L929 cells and responses to both GABA and drugs were assessed using the whole-cell patch clamp technique. Drug potentiation and direct activation were significantly enhanced by mutations in this Cys-loop but these effects were subunit-dependent. Currents in response to agonists were larger when mutations were carried in the alpha and beta subunits but not in the gamma subunit. In contrast, potentiation of current responses by diazepam, etomidate and pentobarbital were all enhanced when mutations were carried in the alpha and gamma subunits, but not the beta subunit. Since the disruption of interactions mediated through the ArProAl motif enhances the mutant receptor's response to both agonist and drugs we suggest that this motif in the Cys-loop of the wild-type receptor participates in interactions that create activation barriers to conformational changes during channel gating.

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