Tsujikawa Hiroto, Song Yumei, Watanabe Makino, Masumiya Haruko, Gupte Sachin A, Ochi Rikuo, Okada Takao
Department of Physiology, New York Medical College, Valhalla, NY 10595, USA.
Am J Physiol Heart Circ Physiol. 2008 Jan;294(1):H285-92. doi: 10.1152/ajpheart.00824.2007. Epub 2007 Nov 2.
Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-beta-cyclodextrin (MbetaCD), we studied the role of cholesterol in the modulation of L-type Ca(2+) currents (I(Ca,L)). MbetaCD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM MbetaCD for 10 min, a positive shift in membrane potential at half-maximal activation (V(0.5)) from -8 to -2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased I(Ca,L) approximately threefold and caused a negative shift in V(0.5) in control cells, but it did not increase I(Ca,L) in MbetaCD-treated myocytes, nor did it shift V(0.5). The effect of MbetaCD (10 or 30 mM) was concentration dependent: 30 mM MbetaCD suppressed the ISO-induced increase in I(Ca,L) more effectively than 10 mM MbetaCD. MbetaCD dialysis also abolished the increase in I(Ca,L) elicited by forskolin or dibutyryl cAMP, but not that elicited by (-)BAY K 8644. External application of MbetaCD-cholesterol complex to rat myocytes attenuated the MbetaCD-mediated inhibition of the ISO-induced increase of I(Ca,L). Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by MbetaCD and increased by MbetaCD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal I(Ca,L) and beta-adrenergic cAMP/PKA-mediated increases in I(Ca,L). We suggest that cholesterol affects the structural coupling between L-type Ca(2+) channels and adjacent regulatory proteins.
胆固醇是质膜的主要成分，包括脂筏/小窝，多种G蛋白偶联受体与信号蛋白和通道在此共定位。通过使用甲基-β-环糊精（MβCD）处理兔和大鼠心室肌细胞中的胆固醇，我们研究了胆固醇在调节L型钙电流（I(Ca,L)）中的作用。在全细胞钳制过程中，MβCD主要从含BAPTA的微电极溶液中透析出来。在用30 mM MβCD透析10分钟的兔心肌细胞中，半数最大激活时的膜电位（V(0.5)）从-8 mV正向偏移至-2 mV，并伴有正电位时电流密度增加（在+20 mV时比时间匹配的对照组增加42%）。异丙肾上腺素（ISO）使对照组细胞的I(Ca,L)增加约三倍，并使V(0.5)负向偏移，但在MβCD处理的心肌细胞中，它并未增加I(Ca,L)，也未使V(0.5)偏移。MβCD（10或30 mM）的作用呈浓度依赖性：30 mM MβCD比10 mM MβCD更有效地抑制ISO诱导的I(Ca,L)增加。MβCD透析还消除了福斯可林或二丁酰cAMP引起的I(Ca,L)增加，但不影响(-)BAY K 8644引起的增加。将MβCD-胆固醇复合物外用于大鼠心肌细胞可减弱MβCD介导的对ISO诱导I(Ca,L)增加的抑制作用。生化分析证实，MβCD使心肌细胞的胆固醇含量降低，而MβCD-胆固醇复合物使其增加。因此，胆固醇似乎有助于调节基础I(Ca,L)以及β-肾上腺素能cAMP/PKA介导的I(Ca,L)增加。我们认为胆固醇影响L型钙通道与相邻调节蛋白之间的结构偶联。
