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Kinetics of Ca²⁺ Binding to the SR Ca-ATPase in the E₁ State

Department of Biology, University of Konstanz, Konstanz, Germany

Correspondence: Address reprint requests to Hans-Jürgen Apell, Dept. of Biology, University of Konstanz, Fach M635, 78457 Konstanz, Germany. Tel.: 49-7531-88-2253; Fax: 49-7531-3183; E-mail: h-j.apell{at}uni-konstanz.de.

The time-resolved kinetics of Ca²⁺ binding to the SR Ca-ATPase in the E₁ state was investigated by Ca²⁺-concentration jump experiments. Ca²⁺ was released by an ultraviolet-light flash from caged calcium, and charge movements in the membrane domain of the ion pumps were detected by the fluorescent styryl dye 2BITC. The partial reaction (H₃E₁ ) E₁ CaE₁ Ca₂E₁ can be characterized by two time constants, ₁ and ₂, both of which are not significantly Ca²⁺-concentration-dependent and only weakly pH-dependent at pH < 7.5. Both time constants differ by a factor of 50 (4.7 vs. 200 ms). The weak substrate-dependence indicates that the rate-limiting process is not related to Ca²⁺ migration through the access channel and ion binding to the binding sites but to conformational rearrangements preceding the ion movements. The high activation energy obtained for both processes, 42.3 kJ mol^–1 and 60.3 kJ mol^–1 at pH 7.2, support this concept. Transient binding of Ca ions to the loop L67 and a movement of the Ca-loaded loop are discussed as a mechanism that facilitates the entrance of both Ca ions into the access channel to the ion-binding sites.

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