1 Smooth muscle cells of the rat portal vein were dispersed by enzymatic treatment and recordings of whole-cell currents under calcium-free conditions were made by the voltage-clamp technique. The effects of the potassium (K)-channel opener, levcromakalim, on K-currents were compared with those of agents which modify protein phosphorylation. 2 Levcromakalim (1-10 muM) added to the extracellular (bath) fluid caused the development of a non-inactivating current (I(K(ATP))) and simultaneously inhibited the delayed rectifier current (I(K(V))) in a concentration-dependent manner. On prolonged exposure to levcromakalim (10 muM), I(K(ATP)) declined and I(K(V)) was further diminished. 3 Addition to the pipette (intracellular) solution of the selective inhibitor of protein kinase C, calphostin C, itself had no effect on K-currents and did not modify the induction of I(K(ATP)) or the simultaneous inhibition of I(K(V)) produced by 1 muM levcromakalim. 4 Addition of the protein kinase inhibitor (PKI(6-22)amide, 1 muM) to the pipette solution caused the production of a glibenclamide-sensitive, non-inactivating current and inhibited I(K(V)). 5 In an assay system, levcromakalim (10 muM) did not inhibit the activity of purified protein kinase A (Type 1 or Type 2). 6 Addition to the pipette solution of the phosphatase inhibitor, okadaic acid (1 muM), did not itself modify K-currents and had little effect on the simultaneous induction of I(K(ATP)) and inhibition of I(K(V)) by levcromakalim (1 muM). 7 When the pipette solution contained 1 mM MgATP (but was depleted of substrates for ATP production), a non-inactivating, glibenclamide-sensitive K-current developed spontaneously in 5 out of 11 cells with the simultaneous reduction of I(K(V)). In 3 of the 6 remaining cells, addition of the dephosphorylating agent, butanedione monoxime (5 mm) to the bath inhibited I(K(V)) and stimulated a glibenclamide-sensitive non-inactivating current. 8 Depletion of intracellular Mg2+ slightly enhanced I(K(V)). Under these conditions, levcromakalim (1 muM and 10 muM) did not significantly induce I(K(ATP)) or inhibit I(K(V)). 9 It is concluded that the effects of levcromakalim on K-currents can be mimicked by procedures designed to reduce channel phosphorylation. The results are consistent with the view that levcromakalim dephosphorylates the delayed rectifier channel, K(V), which becomes converted into a voltage-independent, non-inactivating form known as K(ATP). The possible mechanisms which underlie this interconversion are discussed.
