Four glutamate residues (EEEE locus) are essential for ion selectivity in v
oltage-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 th
is question by examining effects of sulfhydryl-modifying agents (methanethi
osulfonates) on 20 cysteine-substituted mutant forms of an L-type Ca2+ chan
nel. Sulfhydryl modifiers partially blocked whole oocyte Ba2+ currents carr
ied 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 co
valent 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 s
ulfhydryl modification. Sulfhydryl modifiers had lesser effects on channels
substituted one position further from the EEEE locus (+/-2 positions). The
se results indicate that the carboxylate-bearing side chains of the EEEE lo
cus 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 C
a2+ channel selectivity filter differs substantially from that of ancestral
K+ channels.