Levcromakalim may induce a voltage-independent K-current in rat portal veins by modifying the gating properties of the delayed rectifier.
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 microM) added to the extracellular (bath) fluid caused the development of a non-inactivating current (IK(ATP)) and simultaneously inhibited the delayed rectifier current (IK(V)) in a concentration-dependent manner. On prolonged exposure to levcromakalim (10 microM), IK(ATP) declined and IK(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 IK(ATP) or the simultaneous inhibition of IK(V) produced by 1 microM levcromakalim. 4. Addition of the protein kinase inhibitor (PKI(6-22)amide, 1 microM) to the pipette solution caused the production of a glibenclamide-sensitive, non-inactivating current and inhibited IK(V). 5. In an assay system, levcromakalim (10 microM) 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 microM), did not itself modify K-currents and had little effect on the simultaneous induction of IK(ATP) and inhibition of IK(V) by levcromakalim (1 microM). 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 IK(V). In 3 of the 6 remaining cells, addition of the dephosphorylating agent, butanedione monoxime (5 mM) to the bath inhibited IK(V) and stimulated a glibenclamide-sensitive non-inactivating current. 8. Depletion of intracellular Mg2+ slightly enhanced IK(V). Under these conditions, levcromakalim (1 microM and 10 microM) did not significantly induce IK(ATP) or inhibit IK(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 levcromkalim dephosphorylates the delayed rectifier channel, KV, which becomes converted into a voltage-independent, non-inactivating form known as KATP. The possible mechanisms which underlie this interconversion are discussed.
Edwards
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Department of Physiological Sciences, University of Manchester.
Ibbotson
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Weston
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D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology*; D019806:Cromakalim; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000187:drug effects*; D008274:Magnesium / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011169:Portal Vein / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D047428:Protein Kinase Inhibitors; D011758:Pyrroles / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013268:Stimulation, Chemical
D001578:Benzopyrans; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D019806:Cromakalim; C004717:diacetylmonoxime; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D003931:Diacetyl