Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells
Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells
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DOI：
10.1186/1477-7827-7-131
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发表时间：
2009-11-23
影响因子：
4.4
通讯作者：
England, Sarah K.
中科院分区：
文献类型：
--
作者：
Brainard, Adam M.;Korovkina, Victoria P.;England, Sarah K.
背景：大电导、钙和电压激活的钾通道(Maxi-K通道)是决定子宫肌层平滑肌细胞(MSMC)总K+电流的因素之一。该通道响应兴奋性刺激，尤其是细胞内钙离子水平的升高，提供复极化电流，并通过药物手段阻断该通道，诱导MSMCs去极化并增强收缩强度。在MSMC中，MAXI-K通道可以驻留在小窝中，在那里它们与支架蛋白小窝蛋白1(Cav-1)联系在一起。这项研究的目的是研究这种相互作用的后果-更具体地说，破坏Maxi-K通道和Cav-1之间的联系如何影响子宫肌层细胞的当前表达和兴奋性-目的是为了更好地理解子宫功能正常和异常的调节机制。方法：子宫肌层活检收集自择期剖宫产妇女。从这些样本中分离、培养子宫肌细胞，用含有小窝蛋白-1(Cav-1)siRNA或扰乱的Cav-1 siRNA的病毒感染，最后进行膜片钳分析。构建突变的小窝蛋白结合位点MAXI-K通道，并将其导入小鼠成纤维细胞。结果：小窝蛋白-1 siRNA抑制人子宫肌层平滑肌细胞(HMSMC)的总K+电流，与未感染细胞和干扰siRNA对照细胞产生的电流相比明显。Maxi-K通道和小窝蛋白之间的相互作用取决于通道C-末端小窝蛋白结合部位的区域。该位点芳香族残基的突变(突变型F1012A、突变型Y1007A、突变型F1012A和突变型Y1007A、F1012A、Y1015A)导致K+电流比野生型通道导入小鼠成纤维细胞产生的K+电流减少。然而，免疫荧光和免疫沉淀显示，三种芳香族氨基酸(突变体Y1007A、F1012A、Y1015A)的突变是破坏小窝蛋白与Maxi-K通道的联系所必需的。结论：小窝蛋白结合位点的破坏干扰了Cav-1/Maxi-K通道的相互作用，而MSMCs中Cav-1/Maxi-K通道相互作用的缺失会削弱细胞的总K+通道电流。
Background: One determinant of the total K+ myometrial smooth muscle cell (MSMC) current is the large conductance, calcium-and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+, and blocking the channel by pharmacological means induces the depolarization of MSMCs and also enhances contraction strength. In MSMCs, maxi-K channels can reside in the caveolae, where they associate with the scaffolding protein caveolin-1 (cav-1). The aim of this study was to investigate the consequences of this interaction-more specifically, how disruption of the association between the maxi-K channel and cav-1 may influence the current expression and excitability of myometrial cells-with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.Methods: Myometrial biopsies were collected from women undergoing elective C-sections. From these samples, myometrial cells were isolated, cultured, infected with a virus containing either caveolin-1 (cav-1) siRNA or scrambled cav-1 siRNA, and finally subjected to patch-clamp analysis. Mutant caveolin-binding site maxi-K channel constructs were generated and transfected into mouse Ltk-fibroblasts. Channel activity, expression, association, and localization were examined by patch-clamping, Western blot, immunoprecipitation, and immunofluorescence, respectively.Results: The caveolin-1 siRNA suppressed the total K+ current in human myometrial smooth muscle cells (hMSMC), as evident from comparison to the currents generated by both non-infected cells and cells infected with scrambled siRNA controls. The interaction between the maxi-K channel and caveolin depends on a region in the channel's C-terminal caveolin-binding site. Mutations of aromatic residues in this site (mutant F1012A, mutant Y1007A, F1012A and mutant Y1007A, F1012A, Y1015A) resulted in a decrease in K+ current compared to that produced by wild-type channels transfected into mouse Ltk-fibroblasts. However, mutation of all three aromatic amino acids (mutant Y1007A, F1012A, Y1015A) was necessary to disrupt the association between caveolin and the maxi-K channel, as visualized by immunofluorescence and immunoprecipitation.Conclusion: Our results suggest that disruption of the caveolin-binding site interferes with the cav-1/maxi-K channel interaction, and that lack of the cav-1/maxi-K channel interaction in MSMCs attenuates the total K+ channel current of the cell.