Characterization of an amiloride binding region in the α-subunit of ENaC

被引:30
作者
Kelly, O
Lin, CM
Ramkumar, M
Saxena, NC
Kleyman, TR
Eaton, DC
机构
[1] Emory Univ, Sch Med, Dept Physiol, Ctr Cell & Mol Signaling, Atlanta, GA 30322 USA
[2] Emory Univ, Sch Med, Grad Program Biochem Cell & Dev Biol, Grad Div Biol & Biomed Sci, Atlanta, GA 30322 USA
[3] Univ Pittsburgh, Sch Med, Dept Med, Pittsburgh, PA 15261 USA
关键词
open probability; extracellular loop; channel pore; sodium channel; single-channel recording;
D O I
10.1152/ajprenal.00094.2003
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
One of the defining characteristics of the epithelial sodium channel ( ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the alpha-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278 - 283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type alpha-subunits or alpha-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities (P-o) of wild-type channels were distributed into two populations: one with a high P-o and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high-P-o) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type alpha-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.
引用
收藏
页码:F1279 / F1290
页数:12
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