Levcromakalim may induce a voltage‐independent K‐current in rat portal veins by modifying the gating properties of the delayed rectifier

Abstract

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 tIM) 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 gM), 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 gM levcromakalim. 4 Addition of the protein kinase inhibitor (PKI(6-22)amide, 1 JAM) 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 IM) did not inhibit the activity of purified protein kinase A (Type I or Type 2). 6 Addition to the pipette solution of the phosphatase inhibitor, okadaic acid (1 1AM), 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 ylM). 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 JAM and 101 M) 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 levcromakalim dephosphorylates the delayed rectifier channel, Kv, which becomes converted into a voltageindependent, non-inactivating form known as KATP. The possible mechanisms which underlie this interconversion are discussed.

Inhibition of delayed rectifier K+‐current by levcromakalim in single intestinal smooth muscle cells: effects of cations and dependence on K+‐flux

“…In an insulinoma cell line, margatoxin had no effect on IK(DR) but it did inhibit IK(CK) and the ability of (-)Ckm to inhibit IK(DR) . When intracellular Mg2" levels were very low, (-)Ckm did not induce K-current (Beech et al, 1993b) or inhibit IK(DR) (Edwards et al, 1993). Both IK(DR) and A-type K-current were inhibited by KCO drugs in rat portal vein smooth muscle cells (Ibbotson et al, 1993a; Figure 1), suggesting activation of a non-specific mechanism.…”

“…Beech, unpublished observations); (3) (-)Ckm-activated channels and delayed rectifier channels have a similar unitary conductance, in the region of 5 to 20pS with a near-physiological K+-gradient (Beech & Bolton, 1989b;Boyle et al, 1992;Volk & Shibata, 1993;Beech et al, 1993b); (4) a protein kinase inhibitor and a dephosphorylating agent can induce K-current and inhibit IK(DR) (Edwards et al, 1993). If our K+-flux hypothesis is true, observations (1) and (4) can no longer be used to support the conversion hypothesis.…”

“…The effect also occurs with other K channel opener drugs (Ibbotson et al, 1993a) and has been observed in ventricular myocytes (Heath & Terrar, 1994) and in an insulinoma cell line . It is of importance for our understanding of the mechanism of action of levcromakalim because it is the foundation of the 'conversion hypothesis' which proposes that levcromakalimand ATP-sensitive K channels are modulated states of a delayed rectifier K channel (Edwards et al, 1993;Ibbotson et al, 1993b); inhibition of delayed rectifier K-current occurring as delayed rectifier K channels are shifted into a voltage-independent gating mode. If con- ' Author for correspondence.…”

The therapeutic agents that target ATP-sensitive potassium channels

“…These KCOs activate K ATP channels (67)(68)(69). These agents possess high therapeutic potential in treating various clinical conditions such as hypertension, acute and chronic myocardial ischaemia, or congestive heart failure, and also in managing bronchial asthma, urinary incontinence and certain skeletal muscle myopathies (3,45,69).…”

Augmentation by intracellular ATP of the delayed rectifier current independently of the glibenclamide‐sensitive K‐current in rabbit arterial myocytes

“…Both phosphorylation-dependent (Edwards et al, 1993) and independent (Evans et al, 1993) mechanisms have been proposed to regulate these channels. We suggest that the effects of K-channel openers and dephosphorylating conditions on IKDR and IK(ATP) (Edwards et al, 1993) result from independent effects on each channel through inhibition of phosphorylation and/or ATP binding. …”