Pore-lining residues identified by single channel SCAM studies in Cx46 hemichannels
Pore-lining residues identified by single channel SCAM studies in Cx46 hemichannels
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DOI：
10.1080/15419060390264325
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发表时间：
2003-07-01
影响因子：
--
通讯作者：
Verselis, VK
中科院分区：
文献类型：
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作者：
Kronengold, J;Trexler, EB;Verselis, VK
将取代的半胱氨酸可达性方法应用于单个 Cx46 半通道，以鉴定参与衬里由连接蛋白形成的通道的水孔的残基。分配孔的标准包括开放半通道两侧对硫基特异性甲硫磺酸盐 (MTS) 试剂的反应性以及对开放通道性质的可观察影响。我们证明了从 D51 到 L35 的 17 个氨基酸对 MTS 试剂的反应性，这些氨基酸构成了 E1 和 TM1 的片段。定性地，单独的 Cys 取代及其用任一电荷的 MTS 试剂进行修饰所引起的效应的性质表明，侧链价态对于确定单通道特性影响最大，其中 D51 和 L35 分别定义了所识别的孔衬里区域的细胞外和细胞内限制。 TM1 中 L35 以外的许多 Cys 取代导致半通道功能的严重改变并阻止分配到孔中。尽管所有六个亚基都可以通过较小的 MTS 试剂进行修饰，但修饰似乎仅限于使用较大试剂的较少亚基。
The substituted cysteine accessibility method was applied to single Cx46 hemichannels to identify residues that participate in lining the aqueous pore of channels formed of connexins. Criteria for assignment to the pore included reactivity to sulfydryl-specific methanethiosulfonate (MTS) reagents from both sides of an open hemichannel and observable effects on open channel properties. We demonstrate reactivity to MTS reagents over a stretch of seventeen amino acids, D51 through L35, that constitute segments of E1 and TM1. Qualitatively, the nature of the effects caused by the Cys substitutions alone and their modification with MTS reagents of either charge indicate side chain valence is most influential in determining single channel properties with D51 and L35 defining the extracellular and intracellular limits, respectively, of the identified pore-lining region. A number of Cys substitutions beyond L35 in TM1 caused severe alterations in hemichannel function and precluded assignment to the pore. Although all six subunits can be modified by smaller MTS reagents, modifications appear limited to fewer subunits with larger reagents.