title: Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses.
abstract: 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+.
authors.affiliation: The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom. leonard.dode@well.ox.ac.uk
authors.forename: Leonard
authors.initials: L
authors.lastname: Dode
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
authors.forename: Natalie
authors.initials: N
authors.lastname: Leslie
authors.forename: Jittima
authors.initials: J
authors.lastname: Dhitavat
authors.forename: Bente
authors.initials: B
authors.lastname: Vilsen
authors.forename: Alain
authors.initials: A
authors.lastname: Hovnanian
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D007644:Darier Disease / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium