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Localization of Kv4.2 and KChIP2 in lipid rafts and modulation of outward K+ currents by membrane cholesterol content in rat left ventricular myocytes.

Authors: Rudakova, E  Wagner, M  Frank, M  Volk, T 
Citation: Rudakova E, etal., Pflugers Arch. 2015 Feb;467(2):299-309. doi: 10.1007/s00424-014-1521-3. Epub 2014 May 6.
Pubmed: (View Article at PubMed) PMID:24793047
DOI: Full-text: DOI:10.1007/s00424-014-1521-3

Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K(+)-channel alpha-subunit Kv4.2 and the beta-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 degrees 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 muM 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.

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CRRD Object Information
CRRD ID: 10047216
Created: 2015-07-11
Species: All species
Last Modified: 2015-07-11
Status: ACTIVE



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RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.