Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its -adrenergic enhancement in ventricular myocytes.
Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its -adrenergic enhancement in ventricular myocytes.
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DOI：
10.1152/ajpheart.00824.2007
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
H. Tsujikawa;Y. Song;Makino Watanabe;H. Masumiya;S. Gupte;R. Ochi;T. Okada
中科院分区：
文献类型：
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作者：
H. Tsujikawa;Y. Song;Makino Watanabe;H. Masumiya;S. Gupte;R. Ochi;T. Okada
胆固醇是质膜的主要成分，包括脂筏/小窝，在那里各种G蛋白偶联受体与信号蛋白和通道共存。通过甲基-β-环糊精(MbetaCD)对兔和大鼠心室肌细胞胆固醇的调控，研究了胆固醇对L钙电流(I(Ca，L))的调节作用。在全细胞钳制过程中，MbetaCD主要从含BAPTA的移液管溶液中透析出来。在用30 mM MbetaCD透析10分钟的兔心肌细胞中，膜电位从-8 mV正移到-2 mV(V(0.5))，并与正电位下的电流密度增加有关(+20 mV时为42%，与时间匹配的对照组相比)。异丙肾上腺素使对照细胞的I(Ca，L)增加近3倍，V(0.5)负移，但对MbetaCd处理的心肌细胞，它不增加I(Ca，L)，也不使V(0.5)移位。MbetaCd(10或30 mM)的作用呈浓度依赖性：30 mM MbetaCd比10 mM MbetaCd更有效地抑制ISO诱导的细胞内钙离子浓度升高。MbetaCD透析也可阻断Forskolin或二丁酰cAMP引起的i(Ca，L)升高，但不能阻断(-)Bay K8644引起的i(Ca，cAMP)升高。大鼠心肌细胞外用MbetaCD-胆固醇复合体可减弱MbetaCD对异丙肾上腺素引起的心肌细胞内钙升高的抑制作用(L)。生化分析证实MbetaCD降低心肌细胞胆固醇含量，MbetaCD-胆固醇复合体升高心肌细胞胆固醇含量。因此，胆固醇似乎参与了基础i(Ca，L)和β-肾上腺素能cAMP/PKA介导的i(Ca，L)增加的调节。我们认为胆固醇影响L型钙通道与相邻调控蛋白之间的结构偶联。
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.