A Long QT Mutation Substitutes Cholesterol for Phosphatidylinositol-4,5-Bisphosphate in KCNQ1 Channel Regulation

Abstract

IntroductionPhosphatidylinositol-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.Methods and ResultsCOS-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.ConclusionsWe 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.

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

“…Mutant S546L was an exception and displayed a similar sensitivity to Dr-VSP as WT I KS , suggesting that it does not appear to interact functionally with PIP 2 . However, it is possible that, like mutant R539W, it interacts with cholesterol (Coyan et al, 2014).…”

Regulation of KCNQ/Kv7 family voltage-gated K + channels by lipids

“…Indeed in the case of KCNQ1, there is already evidence for additional modes of lipid regulation of function beyond those treated here, including modulation by sphingolipids [165], by cholesterol [150, 166, 167], and by association with lipid rafts [168, 169]. As is often the case in science, advances in technologies that help us understand and explain the world result in an expansion of the degree to which the situation is recognized to be almost unfathomably complex.…”

“…Finally, it should be recognized that the large cytosolic C-terminus of Kv7.1 is thought to contain at least one and maybe more PIP 2 binding sites per subunit [149, 150]. The additional PIP 2 binding events associated with the C-terminus likely collude with other channel-regulatory events such as phosphorylation, association with calmodulin, binding of other ligands, etc.…”

HIV-Tat induces a decrease in I Kr and I Ks via reduction in phosphatidylinositol-(4,5)-bisphosphate availability

“…Again, WT-Tat but not W11Y-Tat led to the same effects as when the channel interaction with PIP 2 is altered ( Figure 1E-H, Table 1). To confirm the hypothesis that Tat reduces the current amplitude of delayed rectifier currents through a decrease in available PIP 2 , we tested the effect of Tat on the mutant KCNE1-R539W-KCNQ1 channel that is insensitive to PIP 2 depletion, when triggered by wortmannin application or by CiVSP phosphatase activation [31]. We compared current amplitude in cells expressing KCNE1-R539W-KCNQ1 in the absence and presence of Tat ( Figure 2).…”