Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation.
Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation.
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氨基末端对 K2P10.1 (TREK-2) 单通道电导的控制：替代翻译起始的作用。
DOI：
10.1113/jphysiol.2008.161927
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Kim,Donghee
中科院分区：
文献类型：
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作者：
Simkin,Dina;Cavanaugh,EricJ;Kim,Donghee
在哺乳动物细胞中表达的TREK-2表现出小（约52 pS）和大（约220 pS）单位电导水平。在这里，我们测试了N末端（69个氨基酸长）在控制单位电导中的作用，以及替代翻译起始作为产生显示不同电导水平的TREK-2亚型的机制的作用。N端前半部分（Δ1-36）的缺失没有影响。然而，大部分N末端（Δ1-66）的缺失导致仅出现大电导通道（Δ 220 pS）。为了支持N末端远侧半部分的关键功能，缺失突变体Δ1-44和Δ1-54分别产生188 pS和190 pS通道。在蛋白质印迹分析中，TREK-2抗体从表达野生型TREK-2的细胞中检测到两条位于约54 kDa和约60 kDa的免疫反应性条带，野生型TREK-2在N末端具有三个潜在的翻译起始位点（指定为M1 M2 M3）。第二个和第三个起始位点从Met突变为Leu（M1 L2 L3）仅产生约60 kDa亚型和小电导通道（约52 pS）。设计用于从第二（M2 L3）或第三（M3）起始位点产生翻译的突变体产生了1054 kDa亚型和大电导通道（10185 -224 pS）。M1 L2 L3、M2 L3和M3对K+具有相对选择性渗透性，这通过[K+]o变化10倍后逆转电位的51-55 mV偏移来判断。M1 L2 L3（≤ 0.02）、M2 L3（≤ 0.02）和M3（≤ 0.03）的PNa/PK值也相似。花生四烯酸、质子和膜拉伸激活，而二丁酰-cAMP抑制TREK-2的所有三种亚型，表明N-末端的缺失不会消除调节。这些结果表明，小电导和大电导TREK-2通道是交替翻译起始的结果，产生具有长和短N末端的亚型，并且N末端的远侧一半控制单位电导。
TREK‐2 expressed in mammalian cells exhibits small (∼52 pS) and large (∼220 pS) unitary conductance levels. Here we tested the role of the N‐terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK‐2 that show different conductance levels. Deletion of the first half (Δ1–36) of the N‐terminus had no effect. However, deletion of most of the N‐terminus (Δ1–66) resulted in the appearance of only the large‐conductance channel (∼220 pS). In support of the critical function of the distal half of the N‐terminus, the deletion mutants Δ1–44 and Δ1–54 produced ∼90 pS and 188 pS channels, respectively. In Western blot analysis, TREK‐2 antibody detected two immunoreactive bands at ∼54 kDa and ∼60 kDa from cells expressing wild‐type TREK‐2 that has three potential translation initiation sites (designated M1M2M3) within the N‐terminus. Mutation of the second and third initiation sites from Met to Leu (M1L2L3) produced only the ∼60 kDa isoform and the small‐conductance channel (∼52 pS). Mutants designed to produce translation from the second (M2L3) or third (M3) initiation site produced the ∼54 kDa isoform, and the large conductance channel (∼185–224 pS). M1L2L3, M2L3and M3were relatively selectively permeable to K+, as judged by the 51–55 mV shifts in reversal potential following a 10‐fold change in [K+]o.PNa/PKvalues were also similar for M1L2L3(∼0.02), M2L3(∼0.02) and M3(∼0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl‐cAMP inhibited all three isoforms of TREK‐2, indicating that deletion of the N‐terminus does not abolish modulation. These results show that the small and large conductance TREK‐2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N‐termini, and that the distal half of the N‐terminus controls the unitary conductance.