Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation
Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation
复制标题
DOI：
10.1073/pnas.92.26.12046
复制
发表时间：
1995-12-19
影响因子：
11.1
通讯作者：
Sullivan, JM
中科院分区：
文献类型：
--
作者：
Choe, S;Stevens, CF;Sullivan, JM
为了探测离子通道的蛋白质环境，我们通过使用位点特异性诱变用两种不同大小的侧链取代跨膜结构域的结构。我们已经使用色氨酸和丙氨酸作为一个高和低影响的扰动探针，分别取代每个18个连续的残基内推定的第二跨膜段，M2，内向整流钾通道，ROMK 1。我们的基本原理是，由于在特定位置处的这些突变而导致的通道功能的变化将反映改变的侧链的结构环境。然后，每个位置可以被分配到三类环境中的一种，如通过不同水平的扰动分级的：很宽容（具有Trp和Ala取代的通道功能），耐受（用Ala取代保留功能，但用Trp取代不保留功能）和不耐受（Ala或Trp取代破坏功能）。我们将非常耐受的环境确定为脂质化的，耐受的是面向内部的蛋白质，而不耐受的是面向孔隙的。我们观察到一个惊人的有序模式的扰动的所有三个环境类，这一结果表明，M2是一个直的α-螺旋。
To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain, Each position can then be assigned to one of three classes of environments, as graded by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-faring, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes, This result indicates that M2 is a straight alpha-helix.