Rudakova Elena, Wagner Michael, Frank Magdalena, Volk Tilmann
Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraße 6, 91054, Erlangen, Germany.
Pflugers Arch. 2015 Feb;467(2):299-309. doi: 10.1007/s00424-014-1521-3. Epub 2014 May 6.
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.
脂筏是细胞膜上富含胆固醇的微结构域。在此，我们研究形成孔道的钾离子通道α亚基Kv4.2和β亚基KChIP2（其为瞬时外向钾电流（Ito）的基础）在左心室肌细胞脂筏中的定位。此外，我们探讨了膜胆固醇耗竭（使用20 mM甲基-β-环糊精（MBCD））对钾离子外向电流的影响。胆固醇饱和的MBCD（20 mM）用作对照。从Wistar大鼠的左心室游离壁分离出肌细胞。通过蔗糖密度梯度离心，随后进行蛋白质免疫印迹分析全细胞蛋白的Triton X-100（4℃）不溶部分。在低密度组分（脂筏）中部分检测到Kv4.2和KChIP2。MBCD处理（5分钟）导致Kv4.2和KChIP2向高密度组分转移。通过全细胞膜片钳评估钾电流。MBCD处理在5分钟内导致Ito降低29±3％（20.0±1.6 pA pF-1对28.5±2.0 pA pF-1，n = 15，p <0.001，膜片钳电位= 40 mV）。对照溶液导致Ito的降低明显较小（17±3％，p <0.（001，与MBCD相比p <0.01）。MBCD诱导非失活电流成分（Isus）增加38±9％（10.1±0.6 pA pF-1对7.6±0.4 pA pF-1，n = 15，p <0.001）。在对照溶液中未观察到这种效应。Isus的增加对100μM4-氨基吡啶或20 mM四乙铵不敏感，因此不太可能由Kv1.5或Kv2.1介导。总之，在大鼠心室心肌细胞中，一部分Kv4.2和KChIP2定位于脂筏中。膜胆固醇耗竭导致Ito净减少约12％，通道蛋白Kv4.2和KChIP2重新分布，并增加延迟整流电流。
