title: Localization of Kv4.2 and KChIP2 in lipid rafts and modulation of outward K+ currents by membrane cholesterol content in rat left ventricular myocytes.
abstract: Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K(+)-channel α-subunit Kv4.2 and the β-subunit KChIP2, underlying the transient outward K(+) current (I to), in lipid rafts in left ventricular myocytes. Furthermore, we explored the impact of membrane cholesterol depletion (using 20 mM methyl-beta-cyclodextrin (MBCD)) on K(+) outward currents. Cholesterol-saturated MBCD (20 mM) served as control. Myocytes were isolated from the left ventricular free wall of Wistar rats. The Triton X-100 (4 °C) insoluble fraction of whole cell protein was analyzed by sucrose density gradient centrifugation followed by Western blot. Kv4.2 and KChIP2 were partially detected in low-density fractions (lipid rafts). MBCD treatment (5 min) resulted in a shift of Kv4.2 and KChIP2 towards high-density fractions. K(+) currents were assessed by whole-cell patch-clamp. MBCD treatment resulted in a 29 ± 3 % decrease in I to (20.0 ± 1.6pApF(-1) vs. 28.5 ± 2.0pApF(-1), n = 15, p < 0.001, V Pip = 40 mV) within 5 min. Control solution resulted in a significantly smaller reduction in I to (17 ± 3 %, p < 0.001, p < 0.01 compared with MBCD). MBCD induced a 38 ± 9 % increase in the non-inactivating current component (I sus) (10.1 ± 0.6pApF(-1) vs. 7.6 ± 0.4pApF(-1), n = 15, p < 0.001). This effect was absent in control solution. The increase in I sus was not sensitive to 100 μM 4-aminopyridine or 20 mM tetraethylammonium, making a contribution of Kv1.5 or Kv2.1 unlikely. In conclusion, in rat ventricular cardiomyocytes, a fraction of Kv4.2 and KChIP2 is localized in lipid rafts. Membrane cholesterol depletion results in ~12 % net reduction of I to, a redistribution of the channel proteins Kv4.2 and KChIP2 and an increased delayed rectifier current.
authors.affiliation: Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraße 6, 91054, Erlangen, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Rudakova
authors.forename: Michael
authors.initials: M
authors.lastname: Wagner
authors.forename: Magdalena
authors.initials: M
authors.lastname: Frank
authors.forename: Tilmann
authors.initials: T
authors.lastname: Volk
mesh_terms: D000200:Action Potentials; D000818:Animals; D002784:Cholesterol / Q000378:metabolism*; D005260:Female; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D051605:Kv Channel-Interacting Proteins / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D051672:Shal Potassium Channels / Q000378:metabolism*
chemical_list: D051605:Kv Channel-Interacting Proteins; D051672:Shal Potassium Channels; D002784:Cholesterol