The γ-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 (GABA(A)) receptor-associated protein (GABARAP), was previously identified as binding to the intracellular domain of GABA(A) receptors by using the yeast two-hybrid screen. In the present work, immunofluorescent staining and green fluorescent protein-tagged receptor subunits showed that GABARAP is associated with and promotes the clustering of GABA(A) receptors in QT-6 quail fibroblasts. The tubulin-binding motif of GABARAP and the gamma2 subunit of the receptor are required. Disruption of microtubules prevents the clustering in a time-dependent manner. When green fluorescent protein-tagged alpha1 or gamma2 subunit coexpressed with beta2, gamma2L, and GABARAP was used, recordings from visually identified cells revealed that clustered GABA(A) receptor had an EC(50) of about 20 microM, vs. 5.7 microM for the diffuse receptor. Clustered receptors deactivated faster and desensitized slower than the diffuse receptors, because of decrease in the apparent affinity of GABA binding. Different properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasynaptic and synaptic clustered receptors in neurons may be due to the organization of the postsynaptic machinery.