Intracellular control of calcium and potassium currents in cardiac cells

Abstract

The intracellular mechanisms by which cardiac Ca current (ICa) and the delayed outward K current (IK) are modulated during beta-adrenergic or muscarinic stimulation were investigated at the level of both single-channel and whole-cell currents in single ventricular myocytes of guinea-pigs. Superfusion of cells with beta-adrenergic agonist increased the amplitude of whole-cell ICa in a dose-dependent manner. In the single-channel recording, neither the amplitude of elementary current nor the total number of active channels was affected but the number of blank records was markedly reduced resulting in a larger amplitude of the ensemble average current. Intracellular dialysis of cells with cyclic AMP (cAMP) or the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) produced a dose-dependent increase in the amplitude of ICa and IK. A non-hydrolysable ATP analogue, AMP-PNP, reduced whereas ATP gamma S enhanced the effects of beta-agonist on ICa and IK, suggesting an involvement of protein phosphorylation during the enhancement of these currents. The regulatory subunit of cAMP-PK, the heat-stable protein-kinase inhibitor (PKI) and type-1 protein phosphatase antagonized the beta-adrenergic enhancement of ICa and IK, but did not eliminate ICa. Acetylcholine (ACh) reduced the amplitude of ICa when ICa was enhanced by either beta-adrenergic agonist, forskolin or 3-isobutyl-1-methyl-xanthine but did ACh not when ICa was enhanced by intracellular dialysis with cAMP or C subunit, suggesting that muscarinic inhibition occurs at the level of adenylate cyclase. Non-hydrolysable GTP analogue, GMP-PNP, uncoupled both beta-adrenergic and muscarinic modulation of ICa. Pertussis toxin selectively eliminated the effect of ACh on ICa. Based on these results, we concluded that the activities of the Ca channel and the delayed outward K channel are controlled by the action of neurotransmitters, which are mediated by GTP-binding proteins and cAMP-dependent protein phosphorylation. It is suggested that phosphorylation of 'Ca-channel-related protein' leads to an increased open probability without changing the total number of channels or the elementary current amplitude.