Functional Coupling of the γ‐Aminobutyric Acid_B Receptor with Calcium Ion Channel and GTP‐Binding Protein and Its Alteration Following Solubilization of the γ‐Aminobutyric Acid_B Receptor

Abstract

Abstract: The coupling mechanism of the γ‐Aminobutyric acid (GABA) B receptor, one of the subtypes of GABA receptors, with calcium ion channel and GTP‐binding protein was examined using a crude synaptic membrane (P 2 ) fraction from the bovine cerebral cortex and a fraction solubilized with sodium deoxycholate. In the P 2 fraction, [ 3 H]GABA binding to the GABA B receptor was increased significantly by the addition of calcium ion, and this enhancement was accentuated further by calcium ion channel blockers such as nicardipine and diltiazem. In contrast, N ‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), a calmodulin antagonist, did not affect on the calcium ion‐induced enhancement of GABA B receptor binding. These results suggest that the GABA B receptor may be functionally coupled with the calcium ion channel, which exhibits an inhibitory modulation against the receptor. On the other hand, GABA B receptor binding, which was noncompetitively inhibited by guanine nucleotides such as GTP, guanosine 5′‐(3‐ O ‐thio)triphosphate (GTPγS), guanosine 5′‐(β,γ‐imido)triphosphate [Gpp(NH)p], and GDP, was competitively inhibited by (‐)‐baclofen. Although the affinity of (‐)‐baclo‐fen for the GABA B receptor was decreased in the presence of GTP, pretreatment of the P 2 fraction with islet‐activating protein (IAP) eliminated the effect of GTP. In addition, GABA and (‐)‐baclofen induced an increase of GTPase activity in the P 2 fraction, and this increase was also eliminated by treatment with IAP. These results suggest that the GABA B receptor may also be functionally coupled with IAP‐sensitive GTP‐binding protein. Treatment of the P 2 fraction with sodium deoxycholate resulted in the highest solubilization of GABA B receptor among various detergents examined. The solubilization, however, completely eliminated the stimulating effects of calcium ion and calcium ion channel blockers as well as the inhibitory effects of GTP and GTP analogues on GABA B receptor binding. Furthermore, the increase of GTPase activity induced by GABA and (‐)‐baclofen was also eliminated following the solubilization. These results suggest that functional coupling of the GABA B receptor with the calcium ion channel and GTP‐binding protein such as N i or N o may be easily destroyed following the solubilization.