ACh released from vagal nerve terminals opens G protein-gated potassium (KG) channels at nodal cells, thereby leading to bradycardia. One of the characteristics of ACh-induced KG currents in native cardiac myocytes is a time-dependent slow increase in current amplitude during hyperpolarizing voltage steps (1). This feature is referred to as “relaxation”. We previously reported that RGS (regulators of G-protein signaling) proteins are responsible for the relaxation behavior (2, 3). RGS proteins are known to accelerate the GTP hydrolysis of G protein α subunits, and sequestrate free G protein βγ subunits. Thus, the proteins hasten the speed of activation and deactivation of ACh-induced KG current, and cause the slowly developed current response. The voltage-dependent regulation of G protein-signaling underlies the latter response. In the present study, we examined the KG currents elicited by several muscarinic agonists (such as ACh and pilocarpine) in atrial myocytes. For electrophysiological experiments, the myocytes were enzymatically isolated from hearts removed from adult male Wistar rats, which were deeply anesthetized by intraperitoneal injection of a combination anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol). Unlike the relaxation of ACh-induced KG currents, the pilocarpine-induced KG currents were time-dependent slow decrease in current amplitude during hyperpolarizing voltage steps. In order to clarify the molecular mechanism underlying this voltage-dependent response, we reconstituted the KG channels in Xenopus oocytes by expressing Kir3.1 and Kir3.4. In oocytes that express Kir3.1/Kir3.4 with m2 muscarinic receptor, we recorded the pilocarpine-induced KG current which did not show the deactivation and failed to reproduce the current response of myocytes. However, when co-expressed with RGS4, we succeeded to record the voltage-dependent response resembling to cardiac KG current. By expressing truncation mutants of RGS4, we could reproduce the result of our previous study that only RGS domain itself was needed to reproduce the relaxation of ACh-induced KG current (3). However, we found that both N-terminus and RGS domain of RGS4 were essential to reproduce the voltage-dependent response of pilocarpine-induced KG currents. These data suggest that RGS4 proteins are critical for the voltage-dependent response, and the RGS4-induced modulation of G protein-signaling are varied with muscarinic agonists.