Ca2+-Dependent PKC Facilitates Voltage-Dependent Activation of IKs Through Phosphorylation of An Isoform Specific Site on the KCNE1 Subunit

Abstract

Protein kinase C (PKC) regulates heart inotropy and chronotropy in both physiological and pathophysiological states. In human heart, at least 6 PKC isofoms are expressed, with the Ca2+ dependent isoforms (classic PKCs, cPKCs) being the most abundant. However, little is known about the effect of cPKC on heart rhythm and cardiac ion channel regulation. The slow delayed rectifier current (IKs) is one of the main currents responsible for cardiomyocyte repolarization. In this study, we investigated the regulation of human IKs regulation by cPKC.Human IKs channel (KCNQ1 and KCNE1) and α1-adrenergic receptor were co-expressed in HEK293T cells. IKs was measured by conventional whole-cell and perforated patch-clamp techniques. The selective α1-adrenergic agonist phenylephrine (30 μM) activated IKs by both shifting the voltage dependence of activation (V1/2) to the left, ∼ −20 mV, and increasing in the maximal conductance (Gmax), ∼ 175%. Pretreatment with cell-permeable cPKC inhibitory peptide selectively blocked the agonist-induced voltage shift, but not the increase in Gmax. Application of a cell-permeable cPKC activator peptide mimicked the agonist-induced leftward shift in V1/2, and showed no increase in Gmax. A mutation in a putative PKC phosphorylation site in the auxiliary subunit, KCNE1(S102A), abolished the cPKC-mediated voltage shift. Expression of the phosphorylation-mimicking mutant, KCNE1(S102E), produced channels that had a leftward shift in V1/2 compared to KCNE1(S102A). Our data indicate that cPKC phosphorylation of KCNE1(S102) facilitates voltage-dependent activation of IKs. In addition, we showed that a mutation associated with Long QT type1 at the S4-S5 linker of KCNQ1 and associated with high cardiac risk, also abolished cPKC activation of this channel. Our results suggest that KCNE1(S102) phosphorylation is transduced through the KCNQ1(S4-S5) linker to modulate channel voltage sensing and thereby facilitate channel opening.