Localization of pacemaker channels in lipid rafts regulates channel kinetics
Localization of pacemaker channels in lipid rafts regulates channel kinetics
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DOI：
10.1161/01.res.0000127621.54132.ae
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发表时间：
2004-05-28
影响因子：
20.1
通讯作者：
DiFrancesco, D
中科院分区：
文献类型：
--
作者：
Barbuti, A;Gravante, B;DiFrancesco, D
脂筏是富含鞘脂和胆固醇的离散膜亚结构域。在心室肌细胞中，小窝（一种脂筏）的功能是将β-肾上腺素能转导途径的几种蛋白质集中在非常接近的位置。我们研究了HEK细胞中表达的HCN 4通道的亚细胞定位，并研究了这种定位对HEK和兔窦房（SAN）细胞起搏通道特性的影响。我们使用不连续蔗糖梯度和Western印迹分析检测HCN 4蛋白在HEK和SAN细胞中，并发现HCN 4蛋白定位于低密度膜组分与flotillin（HEK）或caveolin-3（SAN），小窝的结构蛋白。通过与甲基-β-环糊精（MbetaCD）孵育的细胞破坏脂筏损害了特定的HCN 4定位。它还使HEK细胞中的HCN 4电流和SAN细胞中的If电流的激活中点分别向正方向移动11.9和10.4 mV。这些后者的影响不是由于基础环核苷酸水平的升高，因为胆固醇消耗治疗没有改变目前的反应，环核苷酸。与增加的I-f一致，MbetaCD处理的SAN细胞显示舒张期去极化斜率（87%）和速率（58%）的大幅增加。我们还发现，脂筏解体后，HCN 4和天然f通道失活的动力学速度较慢。总之，我们的工作表明，起搏器通道本地化的脂筏和脂筏的破坏导致通道重新分布在膜内，并修改其动力学特性。
Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the beta-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin ( HEK) or caveolin-3 ( SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-beta-cyclodextrin (MbetaCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of If in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I-f, MbetaCD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4- and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.