VOLTAGE-GATED Ca2+ channels link changes in membrane potential to the
delivery of Ca2+, a key second messenger for many cellular responses1.
Ca2+ channels show selectivity for Ca2+ over more plentiful ions such
as Na+ or K+ by virtue of their high-affinity binding of Ca2+ within
the pore2-6. It has been suggested that this binding involves four con
served glutamate residues7-10 in equivalent positions in the putative
pore-lining regions of repeats I-IV in the Ca2+ channel alpha1 subunit
. We have carried out a systematic series of single amino-acid substit
utions in each of these positions and find that all four glutamates pa
rticipate in high-affinity binding of Ca2+ or Cd2+. Each glutamate car
boxylate makes a distinct contribution to ion binding, with the carbox
ylate in repeat III having the strongest effect. Some single glutamate
-to-lysine mutations completely abolish micromolar Ca2+ block, indicat
ing that the pore does not possess any high-affinity binding site that
acts independently of the four glutamate residues. The prevailing mod
el of Ca2+ permeation2,3 must thus be modified to allow binding of two
Ca2+ ions in close proximity11,12, within the sphere of influence of
the four glutamates. The functional inequality of the glutamates may b
e advantageous in allowing simultaneous interactions with multiple Ca2
+ ions moving single-file within the pore. Competition among Ca2+ ions
for individual glutamates11,12, together With repulsive ion-ion elect
rostatic interaction2,3, may help achieve rapid flux rates through the
channel2-5.