The coupling of GABA B receptors to G-protein-gated inwardly rectifying potassium (GIRK) channels constitutes an important inhibitory pathway in the brain. Here, we examined the mechanism underlying desensitization of agonist-evoked currents carried by homomeric GIRK2 channels expressed in HEK-293T cells. The canonical GABA B receptor agonist baclofen produced GIRK2 currents that decayed by 57.3±1.4% after 60 s of stimulation, and then deactivated rapidly (time constant of 3.90±0.21 s) upon removal of agonist. Surface labeling studies revealed that GABA B receptors, in contrast to l opioid receptors (MOR), did not internalize with a sustained stimulation for 10 min, excluding receptor redistribution as the primary mechanism for desensitization. Furthermore, heterologous desensitization was observed between GABA B receptors and MOR, implicating downstream proteins, such G-proteins or the GIRK channel. To investigate the G-protein turnover cycle, the non-hydrolyzable GTP analogue (GTPcS) was included in the intracellular solution and found to attenuate desensitization to 38.3±2.0%. The extent of desensitization was also reduced (45.3±1.3%) by coexpressing a mutant form of the Gaq G-protein subunit that has been designed to sequester endogenous RGS proteins. Finally, reconstitution of GABA B receptors with Gao G-proteins rendered insensitive to RGS resulted in significantly less desensitization (28.5±3.2%). Taken together, our results demonstrate that endogenous levels of RGS proteins effectively enhance GABA B receptor-dependent desensitization of GIRK currents.

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