Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: An additional transmembrane region at the N terminus
Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: An additional transmembrane region at the N terminus
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DOI：
10.1073/pnas.93.25.14922
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发表时间：
1996-12-10
影响因子：
11.1
通讯作者：
Toro, L
中科院分区：
文献类型：
--
作者：
Wallner, M;Meera, P;Toro, L
大电导电压和Ca 2+敏感性K（MaxiK）通道的成孔α亚基由β亚基调节，β亚基具有由细胞外环分开的两个跨膜区域。为了研究β亚基调节所需的孔形成α亚基中的结构决定因素，我们在人MaxiK通道和果蝇同源物之间构建了嵌合结构，我们显示其对β亚基调节不敏感，并分析了α亚基的拓扑结构。多重序列比对与疏水性图的比较显示，MaxiK通道α亚基在N末端具有独特的疏水片段（S 0）。该片段是除了通常在电压依赖性离子通道中发现的六个推定的跨膜片段（S1-S6）之外的片段。通过体外翻译实验证明了这种独特的S 0区的跨膜性质。此外，信号序列融合的正常功能表达和体外N-连接的糖基化实验表明S 0导致胞外N末端。因此，我们提出了一个新的模型，其中MaxiK通道在N末端（S 0）有第七个跨膜片段。包括S 0在内的41个N端氨基酸从人MaxiK通道到果蝇同源物的嵌合交换将β亚基调节转移到其他无响应的果蝇通道。这两个独特的S 0区和胞外N端是必要的，这种功能的增益。
The pore-forming alpha subunit of large conductance voltage- and Ca2+-sensitive K (MaxiK) channels is regulated by a beta subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming alpha subunit necessary for beta-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to beta-subunit modulation, and analyzed the topology of the alpha subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel alpha subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate the S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers beta-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.