2 TYPES OF MECHANOSENSITIVE CHANNELS IN THE ESCHERICHIA-COLI CELL-ENVELOPE - SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION
2 TYPES OF MECHANOSENSITIVE CHANNELS IN THE ESCHERICHIA-COLI CELL-ENVELOPE - SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION
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DOI：
10.1016/s0006-3495(93)81044-0
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发表时间：
1993-07-01
影响因子：
3.4
通讯作者：
KUNG, C
中科院分区：
文献类型：
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作者：
SUKHAREV, SI;MARTINAC, B;KUNG, C
机械敏感离子通道（mechanosensitiveionchannels，MSCs）是一种在细菌中能提供快速的细胞调节反应的通道，但其分子结构尚未确定。使用膜片钳技术检查来自大肠杆菌（菌株AW 740）的MSC，（a）在巨大的原生质球中，（B）通过融合天然膜囊泡与asolectin脂质体重建后，或（c）通过将辛基葡萄糖苷溶解的膜提取物重新组装成asolectin脂质体。MSC的活动是相似的，在所有三个制剂，由一个大的非选择性MSC的3-nS电导（在200 mM KCl），高负压激活，和一个小的弱阴离子选择性MSC的1 nS激活较低的负压。这两个通道似乎更敏感的脂质体比在原生质球中的吸力。在溶解的膜提取物的凝胶过滤和重构级分后，在脂质体中回收大MSC和小MSC活性。通过对脂质体中重构的各个组分进行斑片取样测定，柱层析中通道活性峰的位置显示，在非变性条件下，大MSC的表观分子量约为60-80 kDa，小MSC的表观分子量约为200-400 kDa。我们得出结论：（a）大MSC和小MSC是不同的分子实体，（B）两种MSC在层析后在脂质体中起作用的事实强烈表明这些通道是通过脂质双层转导的张力门控的，和（c）洗涤剂的色谱分离-溶解的膜蛋白和随后对重构级分的贴片取样可用于鉴定和分离这些MS通道蛋白。
Mechanosensitive ion channels (MSCs) which could provide for fast osmoregulatory responses in bacteria, remain unidentified as molecular entities. MSCs from Escherichia coli (strain AW740) were examined using the patch-clamp technique, either (a) in giant spheroplasts, (b) after reconstitution by fusing native membrane vesicles with asolectin liposomes, or (c) by reassembly of octylglucoside-solubilized membrane extract into asolectin liposomes. MSC activities were similar in all three preparations, consisting of a large nonselective MSC of 3-nS conductance (in 200 mM KCl) that was activated by high negative pressures, and a small weakly anion-selective MSC of 1 nS activated by lower negative pressures. Both channels appeared more sensitive to suction in liposomes than in spheroplasts. After gel filtration of the solubilized membrane extract and reconstituting the fractions, both large MSC and small MSC activities were retrieved in liposomes. The positions of the peaks of channel activity in the column eluate, assayed by patch sampling of individual fractions reconstituted in liposomes, showed an apparent molecular mass under nondenaturing conditions of about 60-80 kDa for the large and 200-400 kDa for the small MSC. We conclude that (a) the large MSC and the small MSC are distinct molecular entities, (b) the fact that both MSCs were functional in liposomes following chromatography strongly suggests that these channels are gated by tension transduced via lipid bilayer, and (c) chromatographic fractionation of detergent-solubilized membrane proteins with subsequent patch sampling of reconstituted fractions can be used to identity and isolate these MS channel proteins.