The gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP) promotes GABA(A) receptor clustering and modulates the channel kinetics

A microtubule-associated protein, gamma-aminobutyric acid type A (GABAA) re ceptor-associated protein (GABARAP), was previously identified as binding t o the intracellular domain of GABA(A) receptors by using the yeast two-hybr id screen. In the present work, immunofluorescent staining and green fluore scent protein-tagged receptor subunits showed that CABARAP is associated wi th and promotes the clustering of GABAA receptors in QT-6 quail fibroblasts . The tubulin-binding motif of GABARAP and the gamma 2 subunit of the recep tor are required. Disruption of microtubules prevents the clustering in a t ime-dependent manner. When green fluorescent protein-tagged alpha 1 or gamm a 2 subunit coexpressed with beta 2, gamma 2L, and GABARAP was used, record ings from visually identified cells revealed that clustered GABAA receptor had an EC50 of about 20 mu M, vs. 5.7 mu M for the diffuse receptor. Cluste red receptors deactivated faster and desensitized slower than the diffuse r eceptors, because of decrease in the apparent affinity of GABA binding. Dif ferent properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasyn aptic and synaptic clustered receptors in neurons may be due to the organiz ation of the postsynaptic machinery.