C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

International audienceVoltage-gated Na(+) (NaV) channels are key regulators of myocardial excitability, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na(+) current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts

Opposite effects of the S4-S5 linker and PIP2 on voltage-gated channel function: KCNQ1/KCNE1 and other channels

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 (S4S5L) and of the S6 C-terminal part (S6T) 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 S4S5L is acting like a ligand binding to S6T 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 S4S5L, the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated) require PIP2 to function properly, confirming its crucial importance as an ion channel co-factor. This is highlighted in cases in which an altered regulation of ion channels by PIP2 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 (PIP2 and S4-S5L), and assesses their potential physiological and pathophysiological roles

C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts

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 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

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 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

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