title: Roles of Tyr122-hydrophobic cluster and K+ binding in Ca2+ -releasing process of ADP-insensitive phosphoenzyme of sarcoplasmic reticulum Ca2+ -ATPase.
abstract: Tyr(122)-hydrophobic cluster (Y122-HC) is an interaction network formed by the top part of the second transmembrane helix and the cytoplasmic actuator and phosphorylation domains of sarcoplasmic reticulum Ca(2+)-ATPase. We have previously found that Y122-HC plays critical roles in the processing of ADP-insensitive phosphoenzyme (E2P) after its formation by the isomerization from ADP-sensitive phosphoenzyme (E1PCa(2)) (Wang, G., Yamasaki, K., Daiho, T., and Suzuki, H. (2005) J. Biol. Chem. 280, 26508-26516). Here, we further explored kinetic properties of the alanine-substitution mutants of Y122-HC to examine roles of Y122-HC for Ca(2+) release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa(2) increased with increasing lumenal Ca(2+) concentration in the mutants with K(0.5) 110-320 microm at pH 7.3. These lumenal Ca(2+) affinities in E2P agreed with those estimated from the forward and lumenal Ca(2+)-induced reverse kinetics of the E1PCa(2)-E2P isomerization. K(0.5) of the wild type in the kinetics was estimated to be 1.5 mM. Thus, E2P of the mutants possesses significantly higher affinities for lumenal Ca(2+) than that of the wild type. The kinetics further indicated that the rates of lumenal Ca(2+) access and binding to the transport sites of E2P were substantially slowed by the mutations. Therefore, the proper formation of Y122-HC and resulting compactly organized structure are critical for both decreasing Ca(2+) affinity and opening the lumenal gate, thus for Ca(2+) release from E2PCa(2). Interestingly, when K(+) was omitted from the medium of the wild type, the properties of the wild type became similar to those of Y122-HC mutants. K(+) binding likely functions via producing the compactly organized structure, in this sense, similarly to Y122-HC.
authors: Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. kyamasak@asahikawa-med.ac.jp
authors: Kazuo
authors: K
authors: Yamasaki
authors: Guoli
authors: G
authors: Wang
authors: Takashi
authors: T
authors: Daiho
authors: Stefania
authors: S
authors: Danko
authors: Hiroshi
authors: H
authors: Suzuki
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry*; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry*; D002522:Chlorocebus aethiops; D007700:Kinetics; D008954:Models, Biological; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D011188:Potassium / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D000244:Adenosine Diphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium