Wang Xiao-Li, Ye Dan, Peterson Timothy E, Cao Sheng, Shah Vijay H, Katusic Zvonimir S, Sieck Gary C, Lee Hon-Chi
Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Biol Chem. 2005 Mar 25;280(12):11656-64. doi: 10.1074/jbc.M410987200. Epub 2005 Jan 23.
The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca(2+)-activated K+ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs. Immunofluorescence imaging showed co-localization of caveolin-1 and BK channels in the BAEC membrane. Immunoprecipitation and glutathione S-transferase pull-down assay results indicated that caveolin-1 and BK channels are physically associated. However, whole cell patch clamp recordings could not detect BK (iberiotoxin-sensitive) currents in cultured BAECs under baseline conditions, even though the presence of BK mRNA and protein expression was confirmed by reverse transcription-PCR and Western blots. Cholesterol depletion redistributed the BK channels to non-caveolar fractions of BAECs, resulting in BK channel activation (7.3 +/- 1.6 pA/picofarad (pF), n = 5). BK currents were also activated by isoproterenol (ISO, 1 microM, 6.9 +/- 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 microM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 microM) did not inhibit the ISO effects. We have also found that caveolin-1 knockdown by small interference RNA activated BK currents (5.3 +/- 1.4 pA/pF, n = 6). We conclude that: 1) BK channels are targeted to caveolae microdomains in vascular endothelial cells; 2) caveolin-1 interacts with BK channels and exerts a negative regulatory effect on channel functions; and 3) BK channels are inactive under control conditions but can be activated by cholesterol depletion, knockdown of caveolin-1 expression, or ISO stimulation. These novel findings may have important implications for the role of BK channels in the regulation of endothelial function.
血管内皮富含小窝，小窝是特殊的膜微区，有助于特定细胞信号转导过程的整合。我们发现，大电导钙激活钾（BK）通道与牛主动脉内皮细胞（BAECs）中的小窝蛋白 - 1相关联。OptiPrep梯度细胞分级分离表明，BK通道集中在BAECs中富含小窝的部分。免疫荧光成像显示小窝蛋白 - 1和BK通道在BAEC膜中共定位。免疫沉淀和谷胱甘肽S - 转移酶下拉试验结果表明，小窝蛋白 - 1和BK通道在物理上相关联。然而，全细胞膜片钳记录在基线条件下无法检测到培养的BAECs中的BK（iberiotoxin敏感）电流，尽管通过逆转录 - PCR和蛋白质印迹证实了BK mRNA和蛋白表达的存在。胆固醇耗竭使BK通道重新分布到BAECs的非小窝部分，导致BK通道激活（7.3±1.6 pA/皮法（pF），n = 5）。异丙肾上腺素（ISO，1 microM）也可激活BK电流（6.9±2.4 pA/pF，n = 6）。移液管溶液中加入小窝蛋白 - 1支架结构域肽（10 microM）完全消除了ISO对BK通道激活的影响，而加入乱序对照肽（10 microM）则不抑制ISO的作用。我们还发现，小干扰RNA介导的小窝蛋白 - 1敲低可激活BK电流（5.3±1.4 pA/pF，n = 6）。我们得出结论：1）BK通道定位于血管内皮细胞的小窝微区；2）小窝蛋白 - 1与BK通道相互作用并对通道功能发挥负调节作用；3）BK通道在对照条件下无活性，但可被胆固醇耗竭、小窝蛋白 - 1表达敲低或ISO刺激激活。这些新发现可能对BK通道在内皮功能调节中的作用具有重要意义。
