Residues lining the inner pore vestibule of human muscle chloride channels

Abstract

Chloride channels belonging to the ClC family are ubiquitous and participate in a wide variety of physiological and pathophysiological processes. To define sequence segments in ClC channels that contribute to the formation of their ion conduction pathway, we employed a combination of site-directed mutagenesis, heterologous expression, patch clamp recordings, and chemical modification of the human muscle ClC isoform, hClC-1. We demonstrate that a highly conserved 8-amino acid motif (P3) located in the linker between transmembrane domains D2 and D3 contributes to the formation of a wide pore vestibule facing the cell interior. Similar to a previously defined pore region (P1 region), this segment functionally interacts with the corresponding segment of the contralateral subunit. The use of cysteine-specific reagents of different size revealed marked differences in the diameter of pore-forming regions implying that ClC channels exhibit a pore architecture quite similar to that of certain cation channels, in which a narrow constriction containing major structural determinants of ion selectivity is neighbored by wide vestibules on both sides of the membrane.