Ion Interactions in the High-Affinity Binding Locus of a Voltage-Gated Ca²⁺ Channel

The selectivity filter of voltage-gated Ca²⁺ channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca²⁺ channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca²⁺ channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd²⁺, which binds very tightly within the EEEE locus, and Ba²⁺ or Li⁺, which bind less tightly and hence exhibit high flux rates: Cd²⁺ binds to the EEEE locus ∼10⁴× more tightly than does Ba²⁺, and ∼10⁸× more tightly than does Li⁺. For wild-type channels, Cd²⁺ entry into the EEEE locus was 400× faster when Li⁺ rather than Ba²⁺ was the current carrier, reflecting the large difference between Ba²⁺ and Li⁺ in affinity for the EEEE locus. For the substitution mutants, analysis of Cd²⁺ block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba²⁺ vs. Li⁺). The dependence of Cd²⁺ block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.