Nonglutamate pore residues in ion selection and conduction in voltage-gated Ca2+ channels

High-affinity, intrapore binding of Ca2+ over competing ions is the essenti al feature in the ion selectivity mechanism of voltage-gated Ca2+ channels. At the same time, several million Ca2+ ions can travel each second through the pore of a single open Ca2+ channel. How such high Ca2+ flux is achieve d in the face of tight Ca2+ binding is a current area of inquiry, particula rly from a structural point of view. The ion selectivity locus comprises fo ur glutamate residues within the channel's pore. These glutamates make uneq ual contributions to Ca2+ binding, underscoring a role for neighboring resi dues in pore function. By comparing two Ca2+ channels (the L-type alpha(1C) , and the non-L-type alpha(1A)) that differ in their pore properties but on ly differ at a single amino acid position near the selectivity locus, we ha ve identified the amino-terminal neighbor of the glutamate residue in motif III as a determinant of pore function. This position is more important in the function of a,, channels than in alpha(1A) channels, For a systematic s eries of mutations at this pore position in alpha(1C), both unitary Ba2+ co nductance and Cd2+ block of Ba2+ current varied with residue volume. Pore m utations designed to make alpha(1C) more like alpha(1A) and vice versa reve aled that relative selectivity for Ba2+ over K+ depended almost solely on p ore sequence and not channel type. Analysis of thermodynamic mutant cycles indicates that the motif III neighbor normally interacts in a cooperative f ashion with the locus, molding the functional behavior of the pore.