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 (IdK) 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) IdK 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 microM) reduced current runup by 50 and 70%, respectively. Delayed dialysis with PKI reversed runup and inhibited IdK to below initial levels. Forskolin (1 microM) caused a reversible increase in IdK, which was inhibited by 4-AP (5 mM). Isoproterenol (1 microM) reversibly enhanced IdK; the increase was sensitive to propranolol (2 microM) and 4-AP (5 mM) and was prevented by dialysis with PKI (10 microM). IdK 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.

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Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.1.h109 ◽

1996 ◽

Vol 271 (1) ◽

pp. H109-H119 ◽

Cited By ~ 18

Author(s):

E. A. Aiello ◽

O. Clement-Chomienne ◽

D. P. Sontag ◽

M. P. Walsh ◽

W. C. Cole

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Outward Current ◽

Muscle Cells ◽

Delayed Rectifier ◽

Whole Cell ◽

Kinase C

The effect of protein kinase C (PKC) activation on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (IdK) was studied in isolated rabbit portal vein smooth muscle cells by use of standard whole cell voltage clamp. The effects of the phorbol ester, 4 beta-phorbol 12,13-dibutyrate (PdBu, 100 nM) and diacylglycerol analogues, 1,2-dioctanoyl-sn-glycerol (1,2-diC8, 10 microM) and 1,3-dioctanoyl-sn-glycerol (1,3-diC8, 10 microM), on macroscopic whole cell IdK were assessed in myocytes dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 5 mM ATP (20-22 degrees C). Activation of PKC by 1,2-diC8 or PdBu caused a decline in IdK that was reversed with washout of drug. 1,2-diC8 had no effect on outward current present after exposure to 4-AP (20 mM). The inactive analogue, 1,3-diC8, did not affect IdK, but subsequent exposure to the active analogue, 1,2-diC8, caused a marked depression of the current. The inhibition of IdK by 1,2-diC8 was significantly reduced by intracellular dialysis with the inhibitors of PKC, chelerythrine (50 microM) and calphostin C (1 microM). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 mM intracellular BAPTA did not affect the suppression of IdK by 1,2-diC8, indicating the involvement of a Ca(2+)-independent isoform of PKC. This study suggests a novel signal transduction mechanism for inhibition of 4-AP-sensitive IdK involving a phosphotransferase reaction catalyzed by PKC in vascular smooth muscle myocytes.

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Regulation of 4-aminopyridine-sensitive, delayed rectifier K+ channels in vascular smooth muscle by phosphorylation

Biochemistry and Cell Biology ◽

10.1139/o96-048 ◽

1996 ◽

Vol 74 (4) ◽

pp. 439-447 ◽

Cited By ~ 73

Author(s):

W. C. Cole ◽

O. Clément-Chomienne ◽

E. A. Aiello

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Membrane Potential ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Protein ◽

Muscle Cells ◽

Delayed Rectifier ◽

Kinase C

Voltage-gated, delayed rectifier K+ current (KV) that is sensitive to 4-aminopyridine (4AP) block has been identified in all vascular smooth muscle tissues studied to date. These channels conduct outward, hyperpolarizing K+ current that influences resting membrane potential and contributes to repolarization of action potentials. Smooth muscle cells in most arterial resistance vessels regulate Ca2+ influx and contractile tone by low amplitude, tonic changes in membrane potential. Block of KV with 4-aminopyridine leads to contraction and an enhanced myogenic response to increased intravascular pressure. We investigated the modulation of KV currents in isolated, freshly dispersed smooth muscle cells from rabbit portal vein and coronary arteries in whole-cell voltage clamp experiments. Our findings indicate that KV channels are regulated by signal transduction mechanisms involving vasoactive agonists that activate cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). In this paper, the properties and potential function of KV channels in vascular smooth muscle are reviewed. Further, the regulation and potential role of alterations in KV due to β-adrenoceptor agonists, adenylyl cyclase and PKA, as well as angiotensin II, diacylglycerol, and PKC are discussed.Key words: potassium channels, smooth muscle, protein kinase A, protein kinase C, membrane potential.

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