Central role of PKCα in isoenzyme-selective regulation of cardiac transient outward current Ito and Kv4.3 channels.

The transient outward current I(to) is an important determinant of the early repolarization phase. I(to) and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and I(to) by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Gö6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCß, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCß siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent I(to) reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native I(to). These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein.

BACKGROUND AND PURPOSE: Two-pore-domain potassium (K2P) channels mediate potassium background (or 'leak') currents, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Inhibition of K2P currents permits membrane potential depolarization and excitation. As expected for key regulators of excitability, leak channels are under tight control from a plethora of stimuli. Recently, signalling via protein tyrosine kinases (TKs) has been implicated in ion channel modulation. The objective of this study was to investigate TK regulation of K2P channels. EXPERIMENTAL APPROACH: The two-electrode voltage clamp technique was used to record K2P currents in Xenopus oocytes. In addition, K2P channels were studied in Chinese hamster ovary (CHO) cells using the whole-cell patch clamp technique. KEY RESULTS: Here, we report inhibition of human K2P3.1 (TASK-1) currents by the TK antagonist, genistein, in Xenopus oocytes (IC50=10.7 microM) and in CHO cells (IC50=12.3 microM). The underlying molecular mechanism was studied in detail. hK2P3.1 was not affected by genistin, an inactive analogue of genistein. Perorthovanadate, an inhibitor of tyrosine phosphatase activity, reduced the inhibitory effect of genistein. Current reduction was voltage independent and did not require channel protonation at position H98 or phosphorylation at the single TK phosphorylation site, Y323. Among functional hK2P family members, genistein also reduced K2P6.1 (TWIK-2), K2P9.1 (TASK-3) and K2P13.1 (THIK-1) currents, respectively. CONCLUSIONS AND IMPLICATIONS: Modulation of K2P channels by the TK inhibitor, genistein, represents a novel molecular mechanism to alter background K+ currents.