Interaction between the RGS domain of RGS4 with G protein α subunits mediates the voltage‐dependent relaxation of the G protein‐gated potassium channel

Abstract

In native cardiac myocytes, there is a time dependence to the G protein‐gated inwardly rectifying K+ (KG) channel current during voltage steps that accelerates as the concentration of acetylcholine is increased. This phenomenon has been called ‘relaxation’ and is not reproduced in the reconstituted Kir3.1/Kir3.4 channel in Xenopus oocytes. We have shown that RGS4, a regulator of G protein signalling, restores relaxation to the reconstituted Kir3.1/Kir3.4 channel. In this study, we examined the mechanism of this phenomenon by expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and mutants of RGS4 in Xenopus oocytes. RGS4 restored relaxation to KG channels activated by the pertussis toxin (PTX)‐sensitive G protein‐coupled m2‐muscarinic receptor but not to those activated by the Gs protein‐coupled β2‐adrenergic receptor. RGS4 induced relaxation not only in heteromeric KG channels composed of Kir3.1 and Kir3.4 but also in homomeric assemblies of either an active mutant of Kir3.1 (Kir3.1/F137S) or an isoform of Kir3.2 (Kir3.2d). Truncation mutants of RGS4 showed that the RGS domain itself was essential to reproduce the effect of wild‐type RGS4 on the KG channel. The mutation of residues in the RGS domain which interact with the α subunit of the G protein (Gα) impaired the effect of RGS4. This study therefore shows that interaction between the RGS domain and PTX‐sensitive Gα subunits mediates the effect of RGS4 on the agonist concentration‐dependent relaxation of KG channels.

Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K + channels

“…RGS proteins interact with G i/o and G q/11 ␣ subunits to increase the intrinsic GTPase rate of the G␣ subunit (19)(20)(21)(22)(23). Overexpression of RGS4, for example, accelerates the channel-deactivation kinetics and changes other kinetic parameters such that the measured time constants are more consistent with those occurring after stimulation of native channels in atrial cells (17,18,(24)(25)(26). Because this family of channels can be activated by a wide variety of G i/o -coupled G protein-coupled receptors (GPCRs) in heterologous and native conditions, it is important to establish how general such processes are, and it is these issues that we address in the current study.…”

“…RGS proteins interact with G i/o and G q/11 ␣ subunits to increase the intrinsic GTPase rate of the G␣ subunit (19-23). Overexpression of RGS4, for example, accelerates the channel-deactivation kinetics and changes other kinetic parameters such that the measured time constants are more consistent with those occurring after stimulation of native channels in atrial cells (17,18,(24)(25)(26). …”

Cellular modelling: experiments and simulation to develop a physiological model of G-protein control of muscarinic K + channels in mammalian atrial cells

“…RGS proteins regulate G-protein signalling by accelerating GTP hydrolysis, and they may be involved in the development of certain cardiovascular pathologies (Wieland & Mittmann 2003;Semplicini et al 2006;Wieland et al 2007;Hendriks-Balk et al 2008). Modulation of GTP hydrolysis by RGS proteins also underlies a voltage-and time-dependent character of the K ACh current in atrial myocytes known as 'relaxation' (figure 3b; Inanobe et al 2001;Ishii et al 2001Ishii et al , 2002. Relaxation of the K ACh current reflects an increasing suppression of channel open probability during depolarization and a gradual recovery during hyperpolarization.…”

PIP 3 inhibition of RGS protein and its reversal by Ca 2+ /calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K + channel

“…The latter timedependent current change is termed ''relaxation'' and is characteristic for native K G current (15). We recently showed that coexpression of RGS protein was mandatory for reconstituting the relaxation behavior of K G current in Xenopus laevis oocytes (11,12), and we further revealed that this characteristic could be imputable to the facilitation of the action of RGS by depolarization-induced Ca 2ϩ entry and resultant formation of Ca 2ϩ ͞ calmodulin (CaM) in native atrial myocytes (13). The molecular mechanism of how Ca 2ϩ ͞CaM facilitates the action of a RGS protein, however, remains unresolved.…”

“…We recently found that RGS proteins were responsible not only for the acceleration of the deactivation time course upon agonist washout (10), but also for the characteristic gating behavior of G protein-activated inward-rectifier K ϩ channels (K G ) in cardiac atrial myocytes (11)(12)(13). K G channels are directly activated by the ␤␥ subunits released from pertussis toxinsensitive G proteins upon receptor stimulation, and contribute to acetylcholine (ACh)-induced deceleration of heart beat and neurotransmitter-evoked slow inhibitory postsynaptic potentials in neurons (14).…”