Dode Leonard, Andersen Jens Peter, Leslie Natalie, Dhitavat Jittima, Vilsen Bente, Hovnanian Alain
The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
J Biol Chem. 2003 Nov 28;278(48):47877-89. doi: 10.1074/jbc.M306784200. Epub 2003 Sep 15.
Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.
开展了稳态和快速动力学研究，以从功能上表征由人肌浆（内质）网Ca²⁺ - ATP酶2（SERCA2）亚型SERCA2a和SERCA2b以及10种 Darier病（DD）突变体在HEK - 293细胞中异源表达时介导的Ca²⁺转运循环的整体和部分反应。相对于SERCA2a，SERCA2b从E1Ca²⁺解离Ca²⁺的速率降低了10倍（即SERCA2b酶在胞质Ca²⁺位点表现出真正的高亲和力），且去磷酸化速率较低。这些基本的动力学差异解释了SERCA2b对胞质Ca²⁺激活的表观亲和力增加以及催化周转速率降低的原因。相对于SERCA1a，两种SERCA2亚型从E2到E1Ca²⁺转变的速率均降低了2倍。此外，7种DD突变体的表达水平与野生型相似。Gly23→Glu和Ser920→Tyr的表达水平降低了2倍，Gly749→Arg的表达水平降低了10倍。Ca²⁺转运与ATP水解之间的解偶联和/或部分反应速率的变化导致10种突变体中有7种功能缺失：Gly23→Glu（解偶联）、Ser186→Phe、Pro602→Leu和Asp702→Asn（阻断E1≈P（Ca²）到E2 - P的转变）、Cys318→Arg（解偶联且E2 - P到E2转变速率降低3倍）以及Thr357→Lys和Gly769→Arg（缺乏磷酸化）。Pro895→Leu活性降低2倍是由于E1≈P（Ca²）到E2 - P转变速率降低2倍所致。Ser920→Tyr是一种独特的DD突变体，相对于野生型SERCA2b，其分子Ca²⁺转运活性增强。在这种情况下，该疾病可能是低表达水平和/或Ca²⁺亲和力降低以及对腔内Ca²⁺抑制敏感性降低的结果。
