Side chain orientation in the selectivity filter of a voltage-gated Ca2+ channel

Four glutamate residues (EEEE locus) are essential for ion selectivity in voltage-gated Ca2+ channels, with ion-specific differences in binding to the locus providing the basis of selectivity. Whether side chain carboxylates or alternatively main chain carbonyls of these glutamates project into the pore to form the ion-binding locus has been uncertain. We have addressed this question by examining effects of sulfhydryl-modifying agents (methanethiosulfonates) on 20 cysteine-substituted mutant forms of an L-type Ca2+ channel. Sulfhydryl modifiers partially blocked whole oocyte Ba2+ currents carried by wild type channels, but this block was largely reversed with washout. In contrast, each of the four EEEE locus glutamate --> cysteine mutants (6 position) was persistently blocked by sulfhydryl modifiers, indicating covalent attachment of a modifying group to the side chain of the substituted cysteine. Cysteine substitutions at positions immediately adjacent to the EEEE locus glutamates (+/-1 positions) were also generally susceptible to sulfhydryl modification. Sulfhydryl modifiers had lesser effects on channels substituted one position further from the EEEE locus (+/-2 positions). These results indicate that the carboxylate-bearing side chains of the EEEE locus glutamates and their immediate neighbors project into the water-filled lumen of the pore to form an ion-binding locus. Thus the structure of the Ca2+ channel selectivity filter differs substantially from that of ancestral K+ channels.