Molecular dissection of the large mechanosensitive ion channel (MscL) of E-coli: Mutants with altered channel gating and pressure sensitivity
Molecular dissection of the large mechanosensitive ion channel (MscL) of E-coli: Mutants with altered channel gating and pressure sensitivity
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DOI：
10.1007/s002329900212
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发表时间：
1997-05-01
影响因子：
2.4
通讯作者：
Martinac, B
中科院分区：
文献类型：
--
作者：
Hase, CC;LeDain, AC;Martinac, B
为了寻找大肠杆菌大机械敏感离子通道的重要功能结构域，我们将该基因的几个突变体克隆到谷胱甘肽S-转移酶融合蛋白表达系统中。突变的MSCL蛋白在两亲性N-端区有氨基酸添加、替换或缺失，和/或在两亲性中心或亲水性C-端区有缺失。用凝血酶对分离的融合蛋白进行蛋白水解性消化，可得到几乎纯净的重组MSCL蛋白，这些重组蛋白被重组为人工脂质体，并通过膜片钳技术检测其功能。在MSCL的N端添加氨基酸残基不影响通道活性，而N端氨基酸序列的缺失或改变是不耐受的，并导致通道表现出改变的压力敏感性和门控。C末端缺失27个氨基酸导致MSCL蛋白形成类似于野生型的通道，而33个C末端氨基酸缺失则使通道活性消失。同样，MSCL的内部两亲性区域的缺失也取消了活性。根据最近提出的MSCL空间模型，我们的结果表明：(I)N-末端部分参与了压力激活的通道，(Ii)基本的通道功能与两者有关，推测的中央两亲性cr-螺旋部分和六个C-末端残基RKKEEP形成了一个电荷簇，跟随推测的M2膜跨越α-螺旋。
In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic cr-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning alpha-helix.