Abstract
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.
Highlights
Ca2ϩ transport by Ca2ϩ-ATPase includes phosphoenzyme isomerization with luminal Ca2ϩ release
The results suggest that these residues function in Ca2؉ 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, leading to Ca2؉ release
In analysis of the EP isomerization/Ca2ϩ release process E1PCa2 3 E2P with occluded Ca2ϩ (E2PCa2) 3 E2P ϩ 2Ca2ϩ, the E2P species need to be rapidly converted to a stabilized E1PCa2 state
Summary
Ca2ϩ transport by Ca2ϩ-ATPase includes phosphoenzyme isomerization with luminal Ca2ϩ release. Further understanding of Ca2ϩ binding/release processes in the transport cycle and roles of residues involved, especially the dynamic key process E1PCa2 3 E2PCa2 3 E2P ϩ 2Ca2ϩ, is hampered by an inability to determine and detect the bound but deoccluded Ca2ϩ in minute amounts of expressed wild type and mutants obtained from cultured cells. The E1PCa2 formed is very stable, probably because of Ca2ϩ substitution of Mg2ϩ bound at the catalytic Mg2ϩ subsite, as found previously (23, 26 –29), withstanding membrane filtration and extensive washing We applied this new method to the EP isomerization and Ca2ϩ release kinetic processes in alanine substitution mutants of the Tyr122 hydrophobic cluster because they are critical for the Ca2ϩ-released E2P ground state structure (16 –18). The detailed analyses suggest that a possibly stepwise assembly of the residues into the Tyr122 hydrophobic cluster takes place for proper Ca2ϩ handling (namely, deocclusion, affinity reduction, and release) and, Ca2ϩ transport coupled with EP processing
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