Liu Lijun, Askari Amir
Department of Physiology, Pharmacology, Metabolism, and Cardiovascular Sciences, Medical Univ. of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804, USA.
Am J Physiol Cell Physiol. 2006 Oct;291(4):C569-78. doi: 10.1152/ajpcell.00002.2006. Epub 2006 Apr 19.
Previous studies showed the presence of a significant fraction of Na(+)-K(+)-ATPase alpha-subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na(+)-K(+)-ATPase with its signaling partners. Because both alpha- and beta-subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na(+)-K(+)-ATPase, we have examined the relative distribution of two major subunit isoforms (alpha(1) and beta(1)) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na(2)CO(3) or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30-40% of alpha(1)-subunits and 80-90% of beta(1)-subunits. Use of Na(2)CO(3) was shown to inactivate Na(+)-K(+)-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained approximately 75% of total myocyte Na(+)-K(+)-ATPase activity. Sealed isolated caveolae exhibited active Na(+) transport. Confocal microscopy supported the presence of alpha,beta-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na(+)-K(+) fluxes, and the sites where the pumping and signaling functions of Na(+)-K(+)-ATPase are integrated. Preferential concentration of beta(1)-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of alpha(1) and beta(1) in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na(+)-K(+)-ATPase in cardiac caveolae.
先前的研究表明，心肌细胞小窝中存在相当一部分钠钾ATP酶α亚基，这表明钠钾ATP酶与其信号伴侣存在小窝相互作用。由于ATP酶活性需要α和β亚基同时存在，为了阐明小窝钠钾ATP酶的泵功能状态，我们检测了成年大鼠心肌细胞小窝膜和非小窝膜中两种主要亚基异构体（α1和β1）的相对分布。当用高盐（碳酸钠或氯化钾）浓度处理的细胞裂解物通过标准密度梯度程序进行分级分离时，得到的轻小窝膜含有30%-40%的α1亚基和80%-90%的β1亚基。已证明使用碳酸钠会使钠钾ATP酶失活；然而，通过氯化钾程序获得的小窝膜未变性，并且含有约75%的心肌细胞总钠钾ATP酶活性。密封的分离小窝表现出活跃的钠转运。共聚焦显微镜支持小窝中存在α、β亚基，免疫沉淀显示这些亚基与小窝蛋白寡聚体相关联。这些发现表明，心脏小窝内陷是活跃钠钾通量的主要通道，也是钠钾ATP酶泵功能和信号功能整合的位点。小窝中β1亚基的优先富集具有细胞特异性；在新生心肌细胞中也有发现，但在成纤维细胞和A7r5细胞中未发现。心肌细胞早期和晚期内体中α1和β1的不均匀分布表明，两个亚基的内化途径不同，这是活性钠钾ATP酶在心脏小窝中选择性定位的来源。
