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Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

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  • Johannes D Clausen, Danmark
  • Ilse Vandecaetsbeek
  • ,
  • Frank Wuytack, KU Leuven, Belgien
  • Peter Vangheluwe, KU Leuven, Belgien
  • Jens Peter Andersen
The molecular mechanism underlying the characteristic high apparent Ca2+ affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11 amino-acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca2+ transport cycle in mutant and chimeric Ca2+-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca2+ dissociation from Ca2E1. Addition of the SERCA2b tail to SERCA1a slowed Ca2+ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca2E1. The interaction of LE with L7/8 is also important for the low rate of the Ca2E1P-E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca2E1 and Ca2E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2-E1 transition in SERCA2b depend critically on the TM11, but not at all on the LE. This applies irrespective of simultaneous exchange of L7/8, thus indicating that interaction of the TM11 with SERCA2 specific sequence element(s) elsewhere in the structure is critical in the E2/E2P states. Collectively these properties ensure a higher Ca2+ affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side
TidsskriftJournal of Biological Chemistry
Sider (fra-til)39460-39469
Antal sider10
StatusUdgivet - 28 sep. 2012

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