Assembly of a Tyr122-hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity-reduction and Release with Phosphoenzyme Isomerization [Bioenergetics]

October 6th, 2015 by Yamasaki, K., Daiho, T., Danko, S., Suzuki, H.

The mechanism whereby events in and around the catalytic site/head of Ca2+-ATPase effect Ca2+ release to the lumen from the transmembrane helices remains elusive. We have developed a method to determine deoccluded bound Ca2+ by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca2+. The assay is applicable to minute amounts of Ca2+-ATPase expressed in COS-1 cells. It was validated by measuring the Ca2+ binding properties of unphosphorylated Ca2+-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca2+ release process E1PCa2 → E2PCa2 → E2P + 2Ca2+. In wild type, Ca2+ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa2 → E2PCa2) followed by very rapid Ca2+ release. In contrast, with alanine mutants of Leu119 and Tyr122 on cytoplasmic part of second transmembrane helix (M2) and Ile179 on the A domain, Ca2+ release in 10 μM Ca2+ lags EP isomerization indicating the presence of a transient E2P state with bound Ca2+. Results suggest that these residues function in Ca2+ affinity-reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and resulting association with the A and P domains, thus leading to Ca2+ release.
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