IKS Is Activated By Both Ca2+ Dependent And Independent Isoforms Of PKC

Abstract

KCNQ1 is co-assembled with KCNE1 to form IKs, one of the main currents responsible for cardiomyocyte repolarization. Our data shows that IKs is regulated by stimulation of several Gq-coupled receptors both in native and heterologous systems, in a biphasic manner, showing an activation and an inhibition phase. For all receptors tested activation was blocked by the PKC inhibitor calphostin C. Mutation of a putative PKC phosphorylation motif (KCNE1(S102)) decreased 50% of the activation, suggesting phosphorylation of this residue is involved in the effect, but not precluding the contribution of other putative PKC phosphorylation sites present in the KCNQ1 subunit. Agonist-induced activation was observed in the presence and absence of intracellular Ca2+ release, but the extent and kinetics of activation were dependent on intracellular Ca2+ release. These results suggest possible roles for both Ca2+-independent and Ca2+-dependent PKC isoforms. To test for this hypothesis we used cell-permeable PKC activator peptides that specifically activate either the Ca2+-dependent classical PKC isoforms or the Ca2+-independent PKCδ isoform. The activator peptide for classical PKC isoforms significantly activated IKs current (@ 25% at +40mV) in HEK-293 cells within 1 min and shifted the voltage dependence of activation toward negative voltages (@ -60 mV). On the other hand, the PKCδ activator peptide strongly increased the maximal conductance of activation of the channel with slower kinetics (@ 160% at 4 min) without changing the channel voltage dependence. Our results suggest that both Ca2+-dependent and Ca2+-independent isoforms of PKC enhance IKs channel activity after GqPCR stimulation, but each isoform regulates the IKs channel in a distinct fashion, possibly through phosphorylation of different sites in the channel.