CA2-LOCUS FOR HIGH-AFFINITY CA2+ INTERACTIONS( CHANNEL SELECTIVITY ATA SINGLE)

Ca2+ channels display remarkable selectivity and permeability, traditi onally attributed to multiple, discrete Ca2+ binding sites lining the pore. Each of the four pore-forming segments of Ca2+ channel al subuni ts contains a glutamate residue that contributes to high-affinity Ca2 interactions. Replacement of all four P-region glutamates with glutam ine or alanine abolished micromolar Ca2+ block of monovalent current w ithout revealing any additional independent high-affinity Ca2+ binding site. Pairwise replacements of the four glutamates excluded the hypot hesis that they form two independent high-affinity sites. Systematic a lterations of side-chain length, charge, and polarity by glutamate rep lacement with aspartate, glutamine, or alanine weakened the Ca2+ inter action, with considerable asymmetry from one repeat to another. The P- region glutamate in repeat I was unusual in its sensitivity to asparta te replacement but not glutamine substitution. While all four glutamat es cooperate in supporting high-affinity interactions with single Ca2 ions, they also influence the interaction between multiple divalent c ations.