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. 2009 Aug 25;106(34):14681-6.
doi: 10.1073/pnas.0902809106. Epub 2009 Aug 17.

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes

Elise Balse  1 , Saïd El-Haou, Gilles Dillanian, Aurélien Dauphin, Jodene Eldstrom, David Fedida, Alain Coulombe, Stéphane N Hatem
Affiliations

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes

Elise Balse et al. Proc Natl Acad Sci U S A. .

Abstract

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current I(Kur). Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent MbetaCD induced a delayed ( approximately 7 min) increase in I(Kur); the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon MbetaCD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the MbetaCD-induced I(Kur) increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in MbetaCD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effect of cholesterol on potassium currents in adult atrial myocytes. (A) Time courses of the effect of 1% MβCD and 500 μM 4-AP perfusions on IKur and holding current measured in hKv1.5-EGFP transduced AAM. (B) Traces of currents recorded in Tyrode, at steady state effect of 1% MβCD and of 4-AP. (C) IKur densities measured in control Tyrode and MβCD (n = 11). (D) Superimposed traces of inward background and outward currents recorded in control and in LDL-treated myocytes. (E) Current-voltage curves in control (n = 11) and LDL-treated (n = 11) myocytes.
Fig. 2.
Fig. 2.
Cholesterol depletion increases the number of channels in the plasma membrane of atrial myocytes. Sample traces from cell-attached membrane showing that MβCD increases single-channel activity in freshly isolated (A) and hKv1.5-EGFP-expressing myocytes (B). Note that under 4-AP the activity of large conductance channels was unmasked (A). o-n, downward open, level n; on, upward open, level n; c, closed level. Arrows and dotted lines indicate the closed state.
Fig. 3.
Fig. 3.
Cholesterol depletion reduces the mobility and redistributes Kv1.5 channels in atrial myocytes. (A) Sequential images from FRAP experiments obtained in control prebleach (prebl.) and at various times postbleach (postbl.) in hKv1.5-EGFP expressing myocytes treated or not with 1% MβCD for 10 min. Arrows indicate representatives ROI. Bar, 10 μm. (B) Kinetics of hKv1.5 fluorescence recovery in control and MβCD conditions. (C) Mobile fraction (Mf) measured at the end of the FRAP experiment. (D) Deconvoluted images of HA surface staining in cultured AAM treated or not with 1% MβCD. (E) Fluorescence intensity of endogenous Kv1.5 channels, hKv1.5-HA-transfected, and hKv1.5-EGFP-transduced AAM. (F) Filipin-stained cells without and with MβCD-treatment.
Fig. 4.
Fig. 4.
The effect of MβCD on IKur involves Kv channel exocytosis. From left to right: diagram indicating the trafficking steps studied, the time course of IKur and holding current, IKur traces and statistical analysis of control and MβCD. (A) Effect of the SNARE inhibitor N-ethylmaleimide (NEM, 250 μM) applied intracellularly for 10 min before MβCD perfusion (n = 5). (B) Effect of a 6 h incubation with 5 μM brefeldin A (BFA), an inhibitor of Golgi exit, before patch-clamp experiment (n = 5). (C) Effect of 10-min dialysis with a nonhydrolyzable GTP analogue, GTP-γ-S (500 μM) before MβCD perfusion (n = 5). TGN, transGolgi Network; ER, endoplasmic reticulum; CV, coated vesicle; LE, late endosome.
Fig. 5.
Fig. 5.
The Rab11 GTP-ase is responsible for the stimulatory effect of MβCD on IKur in atrial myocytes. (A) Picture of the 2 compartments associated to specific Rab proteins. Current densities obtained in control Tyrode and following 1% MβCD application in myocytes transfected with constitutively active (CA) or dominant negative (DN) Rab4 (B) or Rab11 (C) constructs.
Fig. 6.
Fig. 6.
Rab11 and hKv1.5 channel association is suppressed by cholesterol depletion. (A–L) Deconvolution images of Rab11 (red) and GFP (green) staining in control (A–F) and MβCD treated-(G–L) myocytes. Panels (D–F) and (J–L) are enlarged single planes from the squared regions of the corresponding z-stacks in (A–C) and (G–I). Note that the size of double-stained particles was drastically diminished following MβCD-treatment. (M) Distribution of Rab11-positive particles in function of their size in control (n = 6) and MβCD-treated AAM (n = 10). (Scale bar, 10 μm in A–C and G–I; 2 μm in D–F and J–L; 1 pixel, 0.1 μm.)
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