Sheng S, Li J, McNulty K A, Kieber-Emmons T, Kleyman T R
Department of Medicine, Physiology and Pathology, School of Medicine, University of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2001 Jan 12;276(2):1326-34. doi: 10.1074/jbc.M008117200.
Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.
上皮钠通道（ENaC）在细胞外液容量和血压调节中起关键作用。为研究ENaC孔道区域的结构，测定了引入的半胱氨酸残基对巯基反应性甲硫基磺酸衍生物（（2-氨基乙基）甲硫基磺酸氢溴酸盐（MTSEA）和[（2-（三甲基铵）乙基]甲硫基磺酸溴化物（MTSET））以及对Cd(2+)的敏感性。孔道区域氨基末端部分（αVal(569)-αTrp(582)）内的选定突变体对MTSEA、MTSET或Cd(2+)有反应，表现为全细胞Na(+)电流的刺激或抑制。反应性残基不相邻，被2 - 3个残基隔开，其中取代的半胱氨酸残基对试剂无反应，且排列在α-螺旋的一个面上。MTSET对αS580Cβγ mENaC的激活与通道开放概率的大幅增加有关。在孔道区域的羧基末端部分（αSer(583)-αSer(592)），只有一个突变体（αS583C）被MTSEA、MTSET和Cd(2+)快速、几乎完全阻断，而其他几个突变通道被MTSEA或Cd(2+)部分阻断，但不被MTSET阻断。我们的数据表明，ENaC的外孔由一个α-螺旋形成，随后是一个形成选择性过滤器的延伸区域。此外，我们的数据表明孔道区域参与ENaC的门控。
