EFFECTS OF ANIONS ON THE G-PROTEIN MEDIATED ACTIVATION OF THE MUSCARINIC-K+ CHANNEL IN THE CARDIAC ATRIAL CELL-MEMBRANE - INTRACELLULAR CHLORIDE INHIBITION OF THE GTPASE ACTIVITY OF GK

The effects of various intracellular anions on the G protein (G(K))-mediated activation of the muscarinic K+ (K(ACh)) channel were examined in single atrial myocytes isolated from guinea pig hearts. The patch clamp technique was used in the inside-out patch configuration. With acetylcholine (ACh, 0.5-mu-M) in the pipette, 1-mu-M GTP caused different magnitudes of K(ACh) channel activation in internal solutions containing different anions. The order of potency of anions to induce the K(ACh) channel activity at 0.5-mu-M ACh and 1-mu-M GTP was Cl- greater-than-or-equal-to Br- > 1-. In the SO42- or aspartic acid internal solution, no channel openings were induced by 1-mu-M GTP with 0.5-mu-M ACh. In both the Cl- and SO42- internal solutions (with 0.5-mu-M ACh) the relationship between the concentration of GTP and the channel activity was fit by the Hill equation with a Hill coefficient of approximately 3-4. However, the concentration of GTP at the half-maximal activation (K(d)) was 0.2-mu-M in the Cl- and 10-mu-M in the SO42- solution. On the other hand, the quasi-steady-state relationship between the concentration of guanosine-5'-omicron-(3-thiotriphosphate) and the channel activity did not differ significantly between the Cl- and SO42- solutions; i.e., the Hill coefficient was approximately 3-4 and the K(d) was - 0.06-0.08-mu-M in both solutions. The decay of channel activity after washout of GTP in the Cl- solution was much slower than that in the SO42- solution. These results suggest that intracellular Cl- does not affect the turn-on reaction but slows the turn-off reaction of G(K), resulting in higher sensitivity of the K(ACh) channel for GTP. In the Cl- solution, even in the absence of agonists, GTP (> 1-mu-M) or ATP (> 1 mM) alone caused activation of the K(ACh) channel, while neither occurred in the SO42- solution. These observations suggest that the activation of the K(ACh) channel by the basal turn-on reaction of G(K) or by phosphate transfer to G(K) by nucleoside diphosphate-kinase may depend at least partly on the intracellular concentration of Cl-.