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

<div><p>Introduction</p><p>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.</p><p>Methods and Results</p><p>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.</p><p>Conclusions</p><p>We show for the first time that a mutation may shift the channel interaction with PIP₂ to a preference for cholesterol. This <i>de novo</i> 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.</p></div

R539W is insensitive to wortmannin.

<p>A, representative permeabilized-patch current recordings of WT or mutant channels measured before or after a 63-s wortmannin application (10 µmol/L), and during the voltage-clamp protocol shown. Start-to-start interval = 7 s. B, relative current amplitude of WT or mutant channels measured at the end of the depolarizing step (+80 mV), plotted against time. Current values are normalized to the current level measured before wortmannin application (time 0). These experiments were performed at 35°C in the permeabilized-patch configuration. In this configuration, it has been shown that there is no spontaneous current rundown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093255#pone.0093255-Loussouarn2" target="_blank">[24]</a>. C, mean relative current amplitude of WT or mutant channels measured after a 63-s wortmannin application (n = 5–7). *p<0.05, <i>versus</i> WT.</p

R539W is insensitive to Ci-VSP.

<p>A, representative ruptured-patch current recordings of WT or mutant channels coexpressed with the voltage-dependent membrane phosphatase, Ci-VSP, at the first (t = 0 s) and 9^th (t = 64 s) step of depolarization. These currents were measured during the voltage-clamp protocol shown. Start-to-start interval = 8 s. The +80-mV depolarization also allows Ci-VSP activation. B, relative tail-current amplitude (measured at −40 mV) of WT or mutant channels after a depolarization to +80 mV, plotted against time. Current values are normalized to the current amplitude measured before Ci-VSP activation (time 0). C, mean relative current amplitude of WT or mutant channels measured after a 64-s Ci-VSP activation (n = 6–11). *p<0.05, ***p<0.001 versus WT. ^§R555C already ran down before Ci-VSP activation, due to basal Ci-VSP activity, it is thus assimilated to 0 (n = 18). WT condition without Ci-VSP is shown in (B) and (C).</p

R539W is insensitive to osmolarity.

<p>A, B, superimposed representative permeabilized-patch recordings of WT (A) and R539W (B) KCNE1-KCNQ1 concatemer currents, respectively, measured in hyper-, iso- and hypoosmotic conditions using the voltage protocol shown in the insert. C, D, averaged tail-current density (in pA/pF), V_0.5 (in mV) and τ_deact (in ms) measured at −40 mV after a depolarization step to +80 mV for WT (C) and +120 mV for R539W (D) channel, in hyperosmotic (hyper), control (iso), and hypoosmotic solutions (hypo) (n = 10); same voltage protocol as in A. *p<0.05, ***p<0.001 (one-way ANOVA for repeated measures). Protocol and experimental conditions are similar to those used in our previous study analyzing the osmoregulation of KCNE1-KCNQ1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093255#pone.0093255-Piron1" target="_blank">[14]</a>.</p

PepLook models of the 19-aa sequence surrounding R539 and W539.

<p>The sequences QQARKPYDVR539DVIEQYSQG and QQARKPYDVW539DVIEQYSQG were used to calculate amphipathic 3D structure models in water. A, D, the 99 PepLook models of low energy were clustered into three WT (A) and four R539W (D) lead models on the basis of backbone RMSd<1 Å. Ribbon structures of these lead models are fitted in (A and D) demonstrating their large similarity. B, E best models (Prime), with R539 in CPK for the WT fragment (B) and W539 in CPK for the mutant fragment (E). Both residues are protruding in a pin loop-like structure. C, F, hydrophobicity profiles of the WT and R539W Primes are visualized by the hydrophobic (brown) and hydrophilic (green) isopotential surface (+0.1 kcal/mol) around the molecule. The MHP profiles demonstrate, first, the rather important hydrophilicity of the models, and second, that changing R to W, transforms a very polar protuberance to an apolar one.</p

R539W is poorly sensitive to intracellular magnesium.

<p>A, representative giant-patch current recordings of WT or mutant channels measured after 5 and 25-s Mg²⁺ application (1.1 mmol/L free Mg²⁺), and during the voltage-clamp protocol shown. Start-to-start interval = 5 s. B, relative tail-current amplitude (measured at −40 mV) of WT or mutant channels after a depolarization to +80 mV, plotted against time. Current values are normalized to the current level measured before magnesium application (time 0). C, rundown time constant (τ) of WT and mutant channel currents (n = 8–12). *p<0.05, **p<0.01, ***p<0.001 versus WT.</p

Relative position of a series of molecules in the membrane.

<p>All molecules are shown at their optimal position in the IMPALA slab after they were systematically tested at every Å across a water/membrane continuous layer. In all plots, the yellow grid represents the membrane center; the purple grid, the averaged lipid head/acyl chain interface, and the pink grid the averaged lipid/water interface. A, PepLook model of the WT (left) and R539W (right) peptides. In both cases, residue 539 is imbedded in the membrane. B, MHP (Molecular Hydrophobicity Potential) profiles of fragments. R539 is responsible for a large hydrophilic (green) protuberance, whereas W539 is a hydrophobic (brown) protuberance in the membrane. C, MEP (molecular electrostatic profile) of the same molecules showing the e-attractivity of the R539 protuberance. D, E and F, same as in A, B and C except that molecules are, from left to right, an extended fluid form of PIP₂, cholesterol, and a fluid form of DOPC. They highlight the relative position of PIP₂ and peptides and the fact that the more hydrophobic cholesterol is embedded in the membranes.</p

Effect of intracellular magnesium on WT, R539W and R539F mutant channels, and after membrane cholesterol depletion.

<p>A, C, E, relative tail-current amplitude (at −40 mV) of WT or mutant channels measured after a depolarization to +80 mV plotted against time during a 1.1 mmol/L free Mg²⁺ application on a giant-patch. Start-to-start interval = 2 s. Current values are normalized to the current level measured before magnesium application (time 0). Cholesterol depletion was induced by 1 hour of 2 mmol/L cyclodextrin (cyclo) or 24 hours of 10 µmol/L triparanol (tripa) pre-treatment. B, D, F, mean rundown time constant (τ) of WT and mutant channels (B), and with and without 2 mmol/L cyclodextrin (D, n = 9–13) or 10 µmol/L triparanol (F, n = 9–15). *p<0.05, **p<0.01 <i>versus</i> WT. DMSO in which triparanol was diluted (E and F) has an effect on rundown kinetics (τ = 15.4±1.6, n = 7 <i>versus</i> τ = 35.0±3.65, n = 15, without and with DMSO pre-treatment respectively, p<0.01).</p

R539W is sensitive to PKA.

<p>A, representative permeabilized-patch current recordings of WT or R539W channels (co-transfected with yotiao) measured during the voltage-clamp protocol shown. B, mean time course of channel activation by 400 µM cpt-cAMP, 10 µM forskolin and 0.2 µM okadaic acid (cAMP) for WT or R539W KCNE1-KCNQ1 concatemer channel tail currents measured at −40 mV after a 1 s depolarization to +80 mV and normalized to the current value before cAMP application (n = 12–13).</p