Phosholipase C-related inactive protein is involved in trafficking of γ2 subunit-containing GABA_A receptors to the cell surface

The subunit composition of GABA_A receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABA_A receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA_A receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABA_A receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [³H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [³H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA_A receptors and GABA_A receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA_A receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABA_A receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABA_A receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.