PKA and PKC partially rescue Long QT type 1 phenotype by restoring channel-PIP2 interactions

Abstract

Long-QT syndrome causes torsade de pointes arrhythmia, ventricular fibrillation, and sudden death. The most commonly inherited form of long-QT syndrome, LQT1, is due to mutations on the potassium channel gene KCNQ1, which forms one of the main repolarizing cardiac K+ channels, IKs. IKs has been shown to be regulated by both β-adrenergic receptors, via protein kinase A (PKA), and by Gq protein coupled receptors (GqPCR), via protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP2). These regulatory pathways were shown to crosstalk, with PKA phosphorylation increasing the apparent affinity of IKs to PIP2. Here we study the effects of LQT1 mutations in putative PIP2-KCNQ1 interaction sites on regulation of IKs by PKA and GqPCR. The effect of the LQT1 mutations on IKs regulation was tested for mutations in conserved, positively charged amino acids, located in four distinct cytoplamic domains of the KCNQ1 subunit: R174C (S2-S3), R243C (S4-S5), R366Q (proximal c-terminus) and R555C (distal c-terminus). Mutations in the c-terminus of IKs (both proximal and distal) enhanced channel sensitivity to changes in membrane PIP2 levels, consistent with a decrease in apparent channel-PIP2 affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP2-depletion and more sensitive to stimulation of PIP2 production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PI5-kinase). In addition, c-terminus mutants showed a potentiated regulation by PKA. On the other hand, for the two cytoplasmic-loop mutations, an impaired activation by PKA was observed. The effects of the mutations on PKC stimulation of the channel paralleled the effects on PKA stimulation, suggesting that both regulatory inputs are similarly affected by the mutations. We tested whether PKC-mediated activation of IKs, similarly to the PKA-mediated activation, can regulate the channel response to PIP2. After PKC activation, channel was less sensitive to changes in membrane PIP2 levels, consistent with an increase in apparent channel-PIP2 affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP2 by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP2 production. Our data indicates that stimulation by PKA and PKC can partially rescue LQT1 mutant channels with weakened response to PIP2 by strengthening channel interactions with PIP2.