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

Acetylcholine (ACh)-induced deceleration of heart beat and the formation of slow inhibitory postsynaptic potentials by various inhibitory neurotransmitters are mediated by G protein-gated inwardly rectifying K+ (KG) channels (Yamada et al. 1998). The cardiac KG channel is composed of Kir3.1 and Kir3.4, while neuronal channels are mainly composed of Kir3.1 and Kir3.2. Both types of channel can be reconstituted by co-expression of their respective Kir3.0 subunits and pertussis toxin (PTX)-sensitive G protein-coupled receptors in Xenopus oocytes (Kubo et al. 1993; Duprat et al. 1994; Krapivinsky et al. 1995; Lesage et al. 1995). Activation, desensitization and deactivation of the reconstituted ACh-induced KG current are accelerated by regulators of G protein signalling (RGS) proteins, thus mimicking to some extent the properties of native KG currents (Doupnik et al. 1997; Saitoh et al. 1997, 1999; Chuang et al. 1998; Herlitze et al. 1999). Therefore, RGS proteins seem to be involved in the physiological control of the KG channel system. One of the characteristic features of ACh-induced KG current in cardiac myocytes is the property of voltage-dependent ‘relaxation’ (Noma & Trautwein 1978; Yamada et al. 1998). The cardiac KG current activated by ACh is composed of instantaneous and time-dependent components. The instantaneous component reflects the open probability of the KG channel at the holding potential. The time-dependent component reflects the gradual increase in channel open probability upon hyperpolarizing voltage steps (Yamada et al. 1998). The ratio between these components varies in an agonist concentration-dependent manner. Increasing the concentration of ACh ([ACh]) increases the proportion of the instantaneous component and decreases the proportion of the time-dependent component which is also accelerated (Fujita et al. 2000). In this way relaxation is a mechanism for reducing the open probability of KG channels at depolarized potentials at low [ACh]. It can therefore be important in the sino-atrial node for slowing the pacemaker depolarization without affecting the action potential configuration. We recently showed that co-expression of RGS4 restored the agonist concentration-dependent relaxation to the reconstituted KG current in Xenopus oocytes, in addition to the acceleration of activation and deactivation, although we could not reproduce the effect of RGS4 on short-term desensitization (Fujita et al. 2000). The induction of relaxation in the reconstituted KG channel is a newly identified effect of RGS proteins and replicates the ACh-induced voltage-dependent relaxation of the native cardiac KG channel. The molecular mechanism of this phenomenon, however, has not yet been determined. In this study, we examined the signalling pathway mediating the effect of RGS4 on KG current relaxation by heterologously expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and RGS4 mutants in Xenopus oocytes. We found that the RGS domain of RGS4 and PTX-sensitive G proteins were required for RGS4-modulation of the kinetics of the KG current.