PHOSPHORYLATION BY PROTEIN-KINASE-A ENHANCES DELAYED RECTIFIER K+ CURRENT IN RABBIT VASCULAR SMOOTH-MUSCLE CELLS

The effect of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activity on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (I-dK) in isolated rabbit portal vein smooth muscle cells was studied via whole cell voltage clamp (20-22 degrees C). A threefold increase in 4-AP-sensitive (5 mM) I-dK was recorded after gaining cell access during dialysis with 5 mM intracellular ATP and internal Ca2+ buffered to a low level with 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Dialysis with the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate (5 mM) or the specific peptide inhibitor of PKA (PKI; 10 mu M) reduced current runup by 50 and 70%, respectively. Delayed dialysis with PKI reversed runup and inhibited I-dK to below initial levels. Forskolin (1 mu M) caused a reversible increase in I-dK, which was inhibited by 4-AP (5 mM). Isoproterenol (1 mu M) reversibly enhanced I-dK; the increase was sensitive to propranolol (2 mu M) and 4-AP (5 mM) and was prevented by dialysis with PKI (10 mu M) I-dK was enhanced over the entire voltage range of activation and associated with a negative shift in reversal potential of net whole cell current, consistent with hyperpolarization of resting membrane potential. The data provide the first evidence for a signal transduction mechanism involving beta-adrenoceptors, adenylate cyclase, and a phosphotransferase reaction mediated by PKA for the regulation of delayed rectifier K+ channels in vascular smooth muscle.