APPS November 2002 Meeting Abstract 1326

Muscle-type chloride channels of the ClC-family appear to be gated by chloride binding to a site in the channel pore. In the present work the ionic selectivity of ClC-0 chloride channel, expressed in HEK 293 cells was probed by patch-clamp recording in the whole-cell mode. The permeability sequence determined from bi-ionic reversal-potential measurements was SCN^- > Cl^- > Br^- > NO₃^- >> Hexanoate > I^- ~ F^- > ClO₄^- = Benzoate > BrO₃^- ~ Formate ~ ClO₃^- ~ Propionate > Glucuronate ~ Glutamate. Several extracellular foreign anions (A^-) were able to block chloride conductance. The half-effective concentration for reduction of outward conductance followed the sequence NO₃^-<SCN^-~ClO₄^-<ClO₃^-<BrO₃^- ~I^- , although currents were measurable in pure solutions of SCN^- and NO₃^-. Various A^- were able to effect shifts of the voltage-dependence of gating, principally by decreasing the closing rate and affecting its voltage dependence. The ability of A^- to shift the voltage dependence of gating to the left in mixtures of A^- and Cl^- coincided with the apparent blocking affinity of A^-. Previous studies of the ClC-1 homologue have demonstrated ClO₄^->Cl^- permeability ¹. We sought to probe the difference in ClO₄^- permeability between ClC-1 and ClC-0 by making mutations in the putative `selectivity centre'². Two amino acids differ between the two selectivity centres. Mutations G483A and M485V in ClC-1 introduced ClO₄^-<Cl^- and ClO₄^-~Cl^- selectivity respectively. The current results support the notion that permeating anions pause briefly at a minimum of two discrete sites in the ClC-0 pore. Occupancy of one of these sites appears to be coupled to gating of the channel, although non-equilibrium effects determine the overall voltage-dependence of gating. Relative differences in the free-energy of ClO₄^-permeation between ClC-0 and ClC-1 channels are accounted for by small differences in the structure of the chloride co-ordinating `selectivity centre'.