Kinetics of Ca²⁺ binding to the SR Ca-ATPase in the E₁ state

¹ Univ. of Konstanz

^* To whom correspondence should be addressed. E-mail: h-j.apell{at}uni-konstanz.de.

Submitted on June 9, 2005
Revised on June 29, 2005
Accepted on 13 July 2005

	Abstract

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 UV-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 (HE₁ ) E₁ CaE₁ Ca₂E₁ can be characterized by two time constants, ₁ and ₂, which both are not significantly Ca²⁺-concentration dependent and only weakly pH dependent at pH < 7.5. Both time constants differ by a factor of about 50 (4.7 ms 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.

Key Words: active ion transport, caged calcium, electrogenicity, fluorescence, styryl dyes

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