GABA Increases both the Conductance and Mean Open Time of Recombinant GABAA Channels Co-expressed with GABARAP

Abstract

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 ...

Protein interactions involving the γ2 large cytoplasmic loop of GABA A receptors modulate conductance

“…4E). The smallest conductance of ϳ25 pS observed in the presence of peptide is similar to the unitary conductance of recombinant ␣␤␥ GABA A receptors (20 -30 pS), that is, receptors expressed in the absence of GABARAP (6,9) (Fig. 4C and Supplementa1 Fig.…”

“…This system allowed the expression of a defined receptor subtype under conditions that either support (ϩGABARAP) or do not permit (ϪGABARAP) the formation of high-conductance GABA A channels (8,9).…”

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

“…For single channel unitary current recordings, we used a step protocol with a holding potential at 0 mV and stepped to Ϫ120 mV to record single channel events in cellattached configuration. The bath solution contained 140 mM (16,17). Single channel current amplitudes were measured directly from and to base line and were only accepted as valid events if their open durations were more than 15 ms (15).…”

Sustained Structural Change of GABAA Receptor-Associated Protein Underlies Long-Term Potentiation at Inhibitory Synapses on a Cerebellar Purkinje Neuron

“…We speculate that functional modulation of GABA A Rs by binding of structurally altered GABARAP is responsible for the RP expression. When GABARAP is expressed with GABA A R composed of ␣1, ␤2/3, and ␥2 subunits in L929 fibroblasts, both single-channel conductance and mean open time of GABA A R are increased (Everitt et al, 2004;Luu et al, 2006). Considering that the majority of GABA A R subunits expressed in a PN are ␣1, ␤2/3, and ␥2, interaction of GABARAP and GABA A R␥2 might upregulate functional properties of GABA A R in a PN.…”

“…Among them, we attended to GABARAP, a linker protein between GABA A R and tubulin (Wang et al, 1999). The number and/or function of surface GABA A R can be regulated by GABARAP in both heterologous expression systems and neurons (Chen et al, 2000(Chen et al, , 2005Nymann-Andersen et al, 2002;Everitt et al, 2004;Leil et al, 2004;Luu et al, 2006). However, implication of GABARAP in synaptic plasticity has not been explored.…”