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Cholesterol Modulates the Recruitment of Kv1.5 Channels from Rab11-Associated Recycling Endosome in Native Atrial Myocytes

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Cholesterol Modulates the Recruitment of Kv1.5 Channels from Rab11-Associated Recycling Endosome in Native Atrial Myocytes Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes Elise Balsea,b, Saïd El-Haoua,b, Gilles Dillaniana,b, Aure´ lien Dauphinc, Jodene Eldstromd, David Fedidad, Alain Coulombea,b, and Ste´ phane N. Hatema,b,1 aInstitut National de la Sante´et de la Recherche Me´dicale, Unite´Mixte de Recherche Scientifique-956, 75013 Paris, France; bUniversite´Pierre et Marie Curie, Paris-6, Unite´Mixte de Recherche Scientifique-956, 75013 Paris, France; cPlate-forme imagerie cellulaire IFR14, 75013 Paris, France; and dDepartment of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 Edited by Lily Y. Jan, University of California, San Francisco, CA, and approved June 19, 2009 (received for review March 17, 2009) Cholesterol is an important determinant of cardiac electrical proper- important role in the regulation of expression of KCNQ1/KCNE1, ties. However, underlying mechanisms are still poorly understood. pacemaker HCN channels and Kv1.5 channels. This process in- Here, we examine the hypothesis that cholesterol modulates the volves several Rab-GTPases (8–10). Rab-GTPases regulate the turnover of voltage-gated potassium channels based on previous trafficking of vesicles between plasma membrane and intracellular observations showing that depletion of membrane cholesterol in- compartments by regulating sorting, tethering and docking of creases the atrial repolarizing current IKur. Whole-cell currents and trafficking vesicles. Rab4, associated with the early endosome (EE), single-channel activity were recorded in rat adult atrial myocytes mediates the fast recycling process while Rab11, linked to the (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and recycling endosome (RE), is involved in slow recycling of proteins expression were studied using fluorescence recovery after photo- back to the cell surface. However, the role of ion channel turnover bleaching (FRAP) and 3-dimensional microscopy. In both native and in cardiac excitability still remains to be investigated. transduced-AAMs, the cholesterol-depleting agent M␤CD induced a Previous studies have reported that membrane cholesterol can delayed (Ϸ7 min) increase in IKur; the cholesterol donor LDL had an modulate the surface expression of potassium channels (11–13). opposite effect. Single-channel recordings revealed an increased For instance, in neonatal rat myocytes, cholesterol depletion causes number of active Kv1.5 channels upon M␤CD application. Whole-cell a fast redistribution of Kv1.5 channels at the plasma membrane recordings indicated that this increase was not dependent on new (12). Kv1.5 channels are the molecular basis of the main repolar- synthesis but on trafficking of existing pools of intracellular channels izing current of the atria, IKur which is involved in the pathophys- whose exocytosis could be blocked by both N-ethylmaleimide and iology of atrial fibrillation (14). Here, we hypothesized that cho- nonhydrolyzable GTP analogues. Rab11 was found to coimmunopre- lesterol depletion and increased membrane translocation of Kv1.5 cipitate with hKv1.5-EGFP channels and transfection with Rab11 channels may underlie the increased IKur (12). Cholesterol can also dominant negative (DN) but not Rab4 DN prevented the M␤CD- act on different stages of the recycling process in various cell types induced IKur increase. Three-dimensional microscopy showed a de- (15–19). crease in colocalization of Kv1.5 and Rab11 in M␤CD-treated AAM. In this study, trafficking and functional expression of Kv1.5 These results suggest that cholesterol regulates Kv1.5 channel ex- channels were investigated in rat adult atrial myocytes (AAM). pression by modulating its trafficking through the Rab11-associated Using single-channel recordings, high spatial resolution 3D- recycling endosome. Therefore, this compartment provides a sub- microscopy and fluorescence recovery after photobleaching membrane pool of channels readily available for recruitment into the (FRAP), we identified a submembrane pool of Kv channels avail- sarcolemma of myocytes. This process could be a major mechanism able for recruitment into the plasma membrane of myocytes upon for the tuning of cardiac electrical properties and might contribute to cholesterol depletion. PHYSIOLOGY the understanding of cardiac effects of lipid-lowering drugs. Results cardiac myocytes ͉ ion channels ͉ membrane lipids ͉ trafficking ͉ Cholesterol Content Regulates Potassium Currents in Myocytes. Per- channel recycling fusion of 1% M␤CD caused a drastic increase in the amplitude of the sustained outward potassium current, IKur, in AAM overex- ipids are important determinants of cardiac excitability. For pressing EGFP-tagged Kv1.5 channels (at ϩ 60 mV: 21.6 Ϯ 5.3 Linstance, free cholesterol is a major lipid class that regulates pA/pF before M␤CD vs. 62.5 Ϯ 10.8 pA/pF upon M␤CD, n ϭ 11, fluidity, curvature, and stiffness of cellular membranes (1). In P Ͻ 0.001) (Fig. 1). This effect occurred at a fixed time of 6–10 min addition, cholesterol can modulate the function of a number of after drug application. Low concentrations of 4-AP (500 ␮mol/L) cardiac ion currents through effects on channel properties (2, 3). inhibited most of the M␤CD-induced current indicating that it was There is also recent clinical and experimental evidence that lipid- mainly underlain by Kv1.5 channels (Fig. 1 A and B). lowering therapy, such as statins, exert anti-arrhythmic effects. In native (nontransduced) myocytes too, 6–10 min of M␤CD However, these pharmacological effects of statins are poorly un- derstood and could involve their pleiotropic effects as well as the Author contributions: E.B., A.C., and S.N.H. designed research; E.B., S.E.-H., G.D., and A.C. reduction of the cholesterol content of cardiac membranes (4, 5). performed research; A.D., J.E., and D.F. contributed new reagents/analytic tools; E.B., Hence, better understanding of mechanisms responsible for cho- S.E.-H., A.C., and S.N.H. analyzed data; and E.B., J.E., D.F., A.C., and S.N.H. wrote the paper. lesterol effects on cardiac electrical properties is of major interest The authors declare no conflict of interest. from both a fundamental and clinical point-of-view. This article is a PNAS Direct Submission. In cardiomyocytes as in other excitable cells, the surface density Freely available online through the PNAS open access option. of functional channels is a dynamically regulated process and results 1To whom correspondence should be addressed at: Unite´Mixte de Recherche Scientifique- from a balance between endocytic and exocytic trafficking pro- 956, Faculte´deMe´ decine Pierre et Marie Curie, Paris-6, 91 boulevard de l’Hoˆpital, 75013 cesses. For instance, the inhibition of the motor protein dynein Paris, France. E-mail: [email protected]. increases potassium current by preventing the retrograde traffick- This article contains supporting information online at www.pnas.org/cgi/content/full/ ing of Kv channels in cardiomyocytes (6, 7). Recycling has an 0902809106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902809106 PNAS ͉ August 25, 2009 ͉ vol. 106 ͉ no. 34 ͉ 14681–14686 Downloaded by guest on September 29, 2021 A80 B C D E 4-AP 500 µM β 1 % M CD 80 *** 15 I (pA/pF) Control 60 10 pA/pF I Kur Control 60 10 holding current 100 ms LDL 40 MβCD 400 s Control 5 40 20 pA/pF 20 LDL 100 ms -120 -100 -80 -60 -40 -20 20 40 60 80 Current density Current density (pA/pF) 4-AP 500 µM 20 Control Vc (mV) Current density (pA/pF) Current density -5 0 +60 mV +60 mV -80 mV 0 0 200 400 600 800 1000 -100 mV -10 Control MβCD Time (s) -80 mV Fig. 1. Effect of cholesterol on potassium currents in adult atrial myocytes. (A) Time courses of the effect of 1% M␤CD and 500 ␮M 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% M␤CD and of 4-AP. (C) IKur densities measured in control Tyrode and M␤CD (n ϭ 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 ϭ 11) and LDL-treated (n ϭ 11) myocytes. application increased IKur, an effect blocked by 500 ␮mol/L 4-AP their compartmentalization (13), we first investigated the effect of (8.4 Ϯ 2.4 pA/pF before M␤CD vs. 43.0 Ϯ 5.8 pA/pF upon M␤CD, cholesterol depletion on Kv1.5 channel mobility using the FRAP n ϭ 4, P Ͻ 0.01). Two time-dependent components of the outward technique. Randomly selected regions of interest (ROI) at the edge current could be analyzed using a biexponential function to identify of myocytes were photobleached, and fluorescence recovery within fast and slow components that correspond to different channel the ROI was monitored over the time by scanning until steady-state populations (20). Interestingly, the fast component most likely was reached (Fig. 3). The mobile fraction was greatly reduced in corresponding to Kv4.x channels was decreased from 16.4 Ϯ 4.0 to 8.6 Ϯ 1.4 pA/pF without any change in its time-dependent inacti- vation (␶fast control: 24.7 Ϯ 2.9 s; ␶fast M␤CD: 20.4 Ϯ 3.4 s; n ϭ 7). As control experiments, AAM were perfused with a mixture of cholesterol and M␤CD in an 8:2 ratio that did not stimulate IKur in either native or transduced myocytes, as previously described (12). To further examine the role of cholesterol in the regulation of potassium currents, native AAM were incubated for 24 h with 50 ␮g/mL low density lipoprotein (LDL), used as a cholesterol donor.
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