PKA and PKC partially rescue Long QT type 1 phenotype by restoring channel-PIP₂interactions

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).

Characterization of a Binding Site for Anionic Phospholipids on KCNQ1

“…There have also been reports that a number of mutations in the long QT syndrome impair channel-PIP 2 interaction but these mutations are widely distributed throughout the protein and we think that they most likely act in an allosteric fashion (32,60). There is one mutation reported in the residues we have identified (R360G) and this leads to a severe loss of function (59).…”

Long QT mutations disrupt IKS regulation by PKA and PIP2 at the same KCNQ1 helix C-KCNE1 interface

“…8C). Interestingly, these two regulation pathways have recently been suggested to undergo cross talk in such a way that KCNQ1 sensitivity to PIP 2 can be modulated by PKA activation (Choveau et al, 2012;Matavel et al, 2010). Consequently, the LQT mutations examined in this study selectively disrupt either one of these two regulatory processes according to their respective location in this important intersubunit interface.…”

“…Others have identified, in both the KCNQ1 and KCNQ2 proximal C-terminus, a cluster of basic residues just downstream of segment 6 (Telezhkin et al, 2013;Thomas et al, 2011;Zhang et al, 2003). In KCNQ1, residues in the segment-2-segment-3 and segment-4-segment-5 linkers have also been proposed to be important for PIP 2 interaction (Matavel et al, 2010;Zaydman et al, 2013). In addition, basic residues have been identified in coiled-coil helix C and have been suggested to be important for PIP 2 interaction (Matavel et al, 2010;Park et al, 2005).…”

“…In KCNQ1, residues in the segment-2-segment-3 and segment-4-segment-5 linkers have also been proposed to be important for PIP 2 interaction (Matavel et al, 2010;Zaydman et al, 2013). In addition, basic residues have been identified in coiled-coil helix C and have been suggested to be important for PIP 2 interaction (Matavel et al, 2010;Park et al, 2005). Notably, in the present study, the LQT1 mutations located in KCNQ1 helix C (R555C, R555H, K557E, R562M), impaired their physical interaction with KCNE1 Cterminus.…”

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

“…As shown in Fig. 2, D-I Both inhibition and activation of KCNQ channels have been reported (13,19,20). Some of these receptor speciesdependent differences in I Ks modulation might be attributed to activation of different branches of downstream G q signaling, e.g.…”