PKA and PKC partially rescue Long QT type 1 phenotype by restoring channel-PIP 2 interactions

Cardiovascular Research Institute, Department of Medicine, University of Rochester, Rochester, NY, USA.
Channels (Austin, Tex.) (Impact Factor: 2.32). 01/2010; 4(1):3-11. DOI: 10.4161/chan.4.1.10227
Source: PubMed


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).

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Available from: Alessandra Matavel, Jun 09, 2014
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    • "Studying the homologous residues in KCNQ2, Brown and colleagues found that the mutation R325A decreased the on-cell, single-channel open probability and the apparent affinity for diC8 PIP2 in excised patches; in contrast, the mutation K327A did not affect the dose response of KCNQ2 (Telezhkin et al., 2013). Lopes and colleagues found that the long QT syndrome-associated mutation R366Q increased the sensitivity of KCNQ1+KCNE1 channels to PIP2 hydrolysis (Matavel et al., 2010). We have recently shown that charge neutralizing and charge reversing mutations of basic residues at the VSD-PGD interface affect the whole cell current amplitude in a manner that is correlated with their effects on apparent PIP2 affinity and PIP2-dependent VSD-PGD coupling. "
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    ABSTRACT: Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.
    Full-text · Article · May 2014 · Frontiers in Physiology
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    • "Regarding KCNQ1, we have shown that type 1 long QT (LQT1) syndrome can be associated with a decrease in KCNQ1-PIP2 interactions provoked by mutations in the S4–S5 linker (R243H) and in the C-terminal domain CTD (R539W and R555C) [10]. This has since been confirmed for mutations R539W and R555C [11], [12]. "
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    ABSTRACT: Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "More recently, Matavel et al. (2010) gave some new insights on KCNQ1 regulation by PKA and PKC. They tested four point mutations of putative PIP2 interaction sites of the channel (R174C, R243C, R366Q, and R555C) and observed that mutations located in the proximal and distal C-terminus (R366Q and R555C, respectively), enhance the channel sensitivity to variations of membrane PIP2 level, suggesting a decrease in the apparent affinity of these mutant channels to PIP2. "
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    ABSTRACT: Voltage-gated potassium (Kv) channels are tetramers, each subunit presenting six transmembrane segments (S1-S6), with each S1-S4 segments forming a voltage-sensing domain (VSD) and the four S5-S6 forming both the conduction pathway and its gate. S4 segments control the opening of the intracellular activation gate in response to changes in membrane potential. Crystal structures of several voltage-gated ion channels in combination with biophysical and mutagenesis studies highlighted the critical role of the S4-S5 linker (S4S5(L)) and of the S6 C-terminal part (S6(T)) in the coupling between the VSD and the activation gate. Several mechanisms have been proposed to describe the coupling at a molecular scale. This review summarizes the mechanisms suggested for various voltage-gated ion channels, including a mechanism that we described for KCNQ1, in which S4S5(L) is acting like a ligand binding to S6(T) to stabilize the channel in a closed state. As discussed in this review, this mechanism may explain the reverse response to depolarization in HCN-like channels. As opposed to S4S5(L), the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP(2)), stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated) require PIP(2) to function properly, confirming its crucial importance as an ion channel cofactor. This is highlighted in cases in which an altered regulation of ion channels by PIP(2) leads to channelopathies, as observed for KCNQ1. This review summarizes the state of the art on the two regulatory mechanisms that are critical for KCNQ1 and other voltage-gated channels function (PIP(2) and S4S5(L)), and assesses their potential physiological and pathophysiological roles.
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