G protein-independent inhibition of GIRK current by adenosine in rat atrial myocytes overexpressing A1 receptors after adenovirus-mediated gene transfer

G protein-activated inwardly rectifying K+ (GIRK) channels, important regulators of membrane excitability in the heart and central nervous system, are activated by interaction with betagamma subunits from heterotrimeric G proteins upon receptor stimulation. In atrial myocytes various endogenous receptors couple to GIRK channels, including the canonical muscarinic M-2 receptor (M(2)AChR) and the A(1) adenosine receptor (A(1)A(1)AdoR). Saturating stimulation of A(1)AdoR in atrial myocytes activates only a fraction of the GIRK current that is activated via M2AChR, which reflects a lower density of A(1)AdoR. In the present study A(1)AdoR were overexpressed by means of adenovirus-mediated gene transfer using green fluorescent protein (GFP) as the reporter. Confirmatory to a previous study, this resulted in an increased sensitivity of macroscopic GIRK current (ACh-activated K+ current I-K<ACh>)) to stimulation by Ado. However, in the majority of GFP-positive myocytes, exposure to Ado at concentrations greater than or equal to 1 mum resulted in activation of I-K(ACh) followed by a rapid inhibition. In those cells a rebound activation of current was recorded upon washout of Ado. The inhibitory component could be recorded in isolation when IK(ACh) Was activated by M(2)AChR-stimulation and brief pulses of Ado were superimposed. In myocytes loaded with GTP-gamma-S, I-K(ACh), irreversibly activated by brief exposure to agonist, was still reversibly inhibited by Ado, suggesting that inhibition is independent of G protein cycling. In myocytes co-transfected with adenoviral vectors encoding A(1)AdoR and GIRK4 subunit, no inhibition of GIRK current by Ado was observed. As acute desensitization of atrial GIRK current, which is reminiscent of the inhibition described here, has been shown to be absent in myocytes overexpressing GIRK4, this suggests that acute desensitization and the novel inhibition might share a common pathway whose target is the GIRK channel complex or its GIRK1 subunit.