W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition

Abstract

Ca+-calmodulin (Ca2+-CaM)-dependent protein kinase II (Ca2+/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca2+-occupied CaM, and KN-93, an inhibitor of Ca2+/CaMKII, on the Kv4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC50 of 92.4 μM. The block was voltage dependent, with an effective electrical distance of 0.18 ± 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of Kv4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 μM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated Kv4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 μM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca2+/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca2+-CaM, as well as a change in activity of Ca2+-CaM-dependent enzymes, including Ca2+/CaMKII.