HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels
HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels
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DOI：
10.1074/jbc.m610978200
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发表时间：
2007-08-03
影响因子：
4.8
通讯作者：
Accili, Eric A.
中科院分区：
文献类型：
--
作者：
Whitaker, Gina M.;Angoli, Damiano;Accili, Eric A.
超极化激活的环核苷酸调节（HCN）“起搏器”通道亚基是整合的膜蛋白，组装为四聚体，在心脏传导组织和神经细胞中形成通道。先前的研究表明，HCN 2和HCN 4通道亚型在哺乳动物细胞中过表达时会发生物理相互作用，但它们是否能够共同组装并形成功能通道仍不清楚。共组装发生超过自组装的程度以及HCN 2-HCN 4异聚体通道是否在天然组织中形成尚不清楚。在这项研究中，我们展示了共同组装的HCN 2和HCN 4在活的中国仓鼠卵巢细胞使用生物发光共振能量转移（BRET 2），一种新的方法研究四聚体的离子通道亚基。结合电生理学和成像方法的结果，BRET 2数据显示HCN 2和HCN 4亚基以相同的偏好自组装和共组装。我们还证明了HCN 2和HCN 4的共定位和正相关的强度在胚胎小鼠心脏免疫组化，以及这些异构体之间的物理相互作用，在大鼠丘脑的免疫共沉淀。总之，这些数据支持天然组织中HCN 2-HCN 4异聚体通道的形成。
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET2), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET2 data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by co-immunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.