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J Biol Chem, Vol. 273, Issue 32, 20134-20143, August 7, 1998

The Cytoplasmic Loop Located between Transmembrane Segments 6 and 7 Controls Activation by Ca²⁺ of Sarcoplasmic Reticulum Ca²⁺-ATPase

Thierry Menguy, Fabienne Corre, Laurence Bouneau, Stéphane Deschamps, Jesper Vuust Møller^**, Philippe Champeil, Marc le Maire, and Pierre Falson

From the  Section de Biophysique des Protéines et des Membranes, DBCM, Commissariat à l'Energie Atomique et CNRS URA 2096, CE Saclay, 91191 Gif sur Yvette Cedex, France and the ^** Danish Biomembrane Research Centre, Department of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark

During active cation transport, sarcoplasmic reticulum Ca²⁺-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca²⁺ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca²⁺-ATPase to Ca²⁺, i.e. on the affinity of the ATPase for Ca²⁺. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca²⁺, as shown by the Ca²⁺ dependence of ATPase phosphorylation from either ATP or P_i. The reduction in Ca²⁺ affinity for phosphorylation from P_i is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca²⁺, as proposed for some of the intramembranous residues essential for Ca²⁺ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca²⁺-ATPase with proteinase K, in the absence or presence of various Ca²⁺ concentrations, leads to Ca²⁺-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca²⁺ concentrations. This implies that these effects are not due to changes in the conformational state of Ca²⁺-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca²⁺ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca²⁺ during the initial steps of Ca²⁺ binding.

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