Residues lining the inner pore vestibule of human muscle chloride channels

Chloride channels belonging to the ClC family are ubiquitous and participat e in a wide variety of physiological and pathophysiological processes. To d efine sequence segments in ClC channels that contribute to the formation of their ion conduction pathway, we employed a combination of site-directed m utagenesis, heterologous expression, patch clamp recordings, and chemical m odification of the human muscle ClC isoform, hClC-1. We demonstrate that a highly conserved 8-amino acid motif (P3) located in the linker between tran smembrane domains D2 and D3 contributes to the formation of a wide pore ves tibule facing the cell interior. Similar to a previously defined pore regio n (P1 region), this segment functionally interacts with the corresponding s egment of the contralateral subunit. The use of cysteine-specific reagents of different size revealed marked differences in the diameter of pore-formi ng regions implying that ClC channels exhibit a pore architecture quite sim ilar to that of certain cation channels, in which a narrow constriction con taining major structural determinants of ion selectivity is neighbored by w ide vestibules on both sides of the membrane.