Dual regulation of cardiac Na+-K+ pumps and CFTR Cl- channels by protein kinases A and C.

Regulation of Na+-K+ pump current (I p) and cystic fibrosis transmembrane conductance regulator (CFTR) Cl– current (I CFTR) by protein kinases A and C (PKA and PKC) was compared under identical experimental conditions by simultaneous measurement of the two currents in guinea-pig ventricular myocytes whole-cell voltage-clamped at 30–32 °C. Membrane current (I) was monitored at a holding potential (V) of –20 mV. I/V relationships were obtained by hyperpolarizing voltage ramps. Phorbol 12,13-dibutyrate (PDBu, 0.1–1 µM) and chelerythrine (10 µM) were used to stimulate and inhibit, respectively, PKC activity. PKA was stimulated by forskolin (4 µM) and inhibited by H-89 (50 µM). At –20 mV, stimulation of PKC by PDBu increased I p to 121–123% of control. Addition of chelerythrine completely reversed this effect. The PDBu-induced augmentation of I p was voltage dependent. The ratio I p(PDBu)/I p(control) increased from 1.10 at –100 mV to ~1.35 at positive membrane potentials. Stimulation of PKA by forskolin also increased I p voltage dependently (128% of control at –20 mV). The effects of PKC and PKA stimulation on I p were additive. The maximum I p observed in the presence of PDBu and forskolin was 141% of control. Application of either H-89 or chelerythrine reversibly decreased I p by 40% and 24%, respectively, suggesting that basal PKA and PKC activities were involved in the regulation of I p. In the presence of H-89, PDBu was unable to increase I p. Likewise, pre-application of chelerythrine abolished the forskolin-induced augmentation of I p. In contrast to I p, I CFTR (measured simultaneously) was absent under basal conditions. Stimulation of PKA by forskolin activated a pronounced I CFTR. Stimulation of PKC by PDBu, on the other hand, neither activated the Cl– current significantly nor increased I CFTR pre-activated by forskolin. Inhibition of PKC by chelerythrine, however, attenuated the PKA-mediated activation of I CFTR. The results reveal a complex interplay between PKA and PKC in regulating cardiac I p and I CFTR with some similarities but also important differences. I p is increased voltage dependently and additively by stimulation of both kinases. The steady-state activity of each of the kinases is involved in the modulation of basal I p and obligatory for the augmentation of I p induced by stimulation of the other kinase. In contrast, there appears to be no basal I CFTR. I CFTR is activated significantly only after stimulation of PKA. PKC activity, however, appears to facilitate this activation.