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Time-Resolved Charge Translocation by Sarcoplasmic Reticulum Ca-ATPase Measured on a Solid Supported Membrane

Francesco Tadini Buoninsegni ^*, Gianluca Bartolommei ^*, Maria Rosa Moncelli ^*, Giuseppe Inesi  and Rolando Guidelli ^*

^* Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Italy; and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 USA

Correspondence: Address reprint requests to Prof. Rolando Guidelli, Dept. of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy. Tel.: 39-055-4573097; Fax: 39-055-4573098; E-mail: guidelli{at}unifi.it.

Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K⁺ ions and at different pH values. Ca²⁺ concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca²⁺ concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca²⁺ concentration, with a half-saturating value of 1.5 µM. The pH dependence of the charge after Ca²⁺ translocation demonstrates the occurrence of one H⁺ per one Ca²⁺ countertransport at pH 7 by direct charge-transfer measurements. The presence of K⁺ decreases the magnitude of the current transients without altering their shape; this decrease is explained by K⁺ binding to the cytoplasmic side of the pump in the E₁ conformation and being released to the same side during the E₁–E₂ transition.

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