Endogenous RGS proteins enhance acute desensitization of GABAB receptor-activated GIRK currents in HEK-293T cells

Abstract

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.

γ-Aminobutyric Acid Type B Receptors Are Constitutively Internalized via the Clathrin-dependent Pathway and Targeted to Lysosomes for Degradation

“…In agreement with previous studies (16,19,20), no agonistinduced internalization of GABA B receptors was observed upon prolonged agonist treatment (10 -120 min), irrespective of the method used. Thus, unlike many other G protein-coupled receptors, GABA B receptors display robust fast constitutive but no agonist-promoted internalization.…”

“…Consistent with the atypical mode of desensitization, recent findings suggest that GABA B receptors are not internalized and do not recruit arrestin in response to agonist exposure (16,19,20). Interestingly, Fairfax et al (19) hypothesized that GABA B receptors may be targeted directly from the surface to the proteasome for degradation.…”

“…A second mechanism proposed involves the interaction of the GABA B receptor heterodimer with N-ethylmaleimide-sensitive fusion protein (NSF), which primes the receptor for phosphorylation by protein kinase C upon agonist stimulation and leads to desensitization (18). Thus, although the mechanism of GABA B receptor desensitization is still poorly understood, it is clearly mediated by processes distinct from the generally accepted model of G protein receptor kinase phosphorylation-induced desensitization and subsequent internalization of the receptors.Consistent with the atypical mode of desensitization, recent findings suggest that GABA B receptors are not internalized and do not recruit arrestin in response to agonist exposure (16,19,20). Interestingly, Fairfax et al (19) hypothesized that GABA B receptors may be targeted directly from the surface to the proteasome for degradation.…”

Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABAB receptors in cortical neurons

“…This finding is in line with so far published data obtained under similar experimental conditions irrespective whether receptors were expressed in neurons (Fairfax et al, 2004;Vargas et al, 2008) or in heterologous expression systems (Fairfax et al, 2004;Grampp et al, 2007;Mutneja et al, 2005;Perroy et al, 2003). These experiments suggest that chronic activation does not promote endocytosis of GABA B receptors.…”

In Vivo Ethanol Experience Increases D2 Autoinhibition in the Ventral Tegmental Area

“…However, blockade of IP 3 -mediated Ca 2 + release failed to affect desensitization of baclofen-induced currents in this study, demonstrating that Ca 2 + -dependent desensitization is selectively involved in the regulation of D 2 , but not GABA B , receptor signaling in dopamine neurons. It has been shown that GABA B receptor-GIRK channel coupling efficiency, as well as agonistinduced desensitization of GABA B receptor-mediated currents, are regulated by regulator of G protein signaling (RGS) proteins (Labouebe et al, 2007;Mutneja et al, 2005). These findings strongly suggest that these two G i/o -coupled receptors are differentially regulated by distinct desensitization mechanisms in dopamine neurons.…”