Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization.

The mechanism whereby events in and around the catalytic site/head of Ca(2+)-ATPase effect Ca(2+) release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca(2+) by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca(2+). The assay is applicable to minute amounts of Ca(2+)-ATPase expressed in COS-1 cells. It was validated by measuring the Ca(2+) binding properties of unphosphorylated Ca(2+)-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca(2+) release process E1PCa2 → E2PCa2 → E2P + 2Ca(2+). In the wild type, Ca(2+) release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa2 → E2PCa2) followed by very rapid Ca(2+) release. In contrast, with alanine mutants of Leu(119) and Tyr(122) on the cytoplasmic part of the second transmembrane helix (M2) and Ile(179) on the A domain, Ca(2+) release in 10 μm Ca(2+) lags EP isomerization, indicating the presence of a transient E2P state with bound Ca(2+). The results suggest that these residues function in Ca(2+) affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca(2+) release.