title: 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 beta-adrenergic receptors, via protein kinase A (PKA), and by Gq protein coupled receptors (GqPCR), via protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). These regulatory pathways were shown to crosstalk, with PKA phosphorylation increasing the apparent affinity of IKs to PIP(2). Here we study the effects of LQT1 mutations in putative PIP(2)-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 PIP(2) levels, consistent with a decrease in apparent channel-PIP(2) affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP(2)-depletion and more sensitive to stimulation of PIP(2) 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 PIP(2). After PKC activation, channel was less sensitive to changes in membrane PIP(2) levels, consistent with an increase in apparent channel-PIP(2) affinity. PKC-activated channel was less sensitive to inhibition caused by block of synthesis of PIP(2) by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of PIP(2) production. Our data indicates that stimulation by PKA and PKC can partially rescue LQT1 mutant channels with weakened response to PIP(2) by strengthening channel interactions with PIP(2).
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, University of Rochester, Rochester, NY, USA.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Matavel
authors.forename: Emiliano
authors.initials: E
authors.lastname: Medei
authors.forename: Coeli M B
authors.initials: CM
authors.lastname: Lopes
mesh_terms: D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D005838:Genotype; D006801:Humans; D015640:Ion Channel Gating; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D009154:Mutation; D009865:Oocytes; D010641:Phenotype; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D029597:Romano-Ward Syndrome / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D013997:Time Factors; D014981:Xenopus
chemical_list: D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate; D043562:Receptors, G-Protein-Coupled; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D043802:GTP-Binding Protein alpha Subunits, Gq-G11