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I.V. Peshenko, A.M. Dizhoor; Identification of Ca2+– and Mg2+– Specific EF–Hands in GCAP–1 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1712.
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Purpose: GCAPs are Ca2+/Mg2+ sensor proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca2+ and inhibit RetGC in a Ca2+ sensitive manner. The range of Ca2+–sensitivity of RetGC regulation is set by free Mg2+ . The purpose of this study was to identify Ca2+/Mg2+ specific EF–hand(s) in GCAP–1. Methods: Recombinant bovine GCAP–1 was expressed in E. coli, and its conformational changes were analyzed by tryptophan fluorescence. Site directed mutagenesis was used to disable EF–hands 2, 3 and 4 by substituting carboxyl–containing side chanes in their cation–binding loops with their amides or with glycin. Results: Ca2+–dependent changes in tryptophan fluorescence of wild–type GCAP–1 are biphasic: the intensity of the fluorescence decreases at 100 nM free Ca2+ (phase I) followed by increase in the fluorescence as Ca2+ rises from above 100 nM (phase II). Mg2+ binding produces phase I but not phase II. We have found that disabling of the EF hand 4 in GCAP–1 (EF4– mutant) eliminates the phase II, but does not affect phase I. We also tested GCAP–1 mutants with only one remaining EF hand (double mutants, EF2/3–, EF3/4– and EF2/4–). No phase I was observed in case of EF2/3– mutant. The EF3/4– mutant exhibited the phase I with half–maximal concentrations of Ca2+ ([Ca]1/2) and Mg2+ ([Mg]1/2) at near 20 nM and 0.4 mM, respectively. The EF2/4– mutant also exhibited only phase I, [Ca]1/2 and [Mg]1/2 of ∼ 2 µM and 8 mM respectively. When both EF–hand 4 and EF–hand 3 remained intact, the [Ca]1/2 and [Mg]1/2 for the EF–hand 3 remained close to ∼100 nM and ∼1 mM, respectively. Conclusions: Both EF–hands 2 and 3 contribute to the phase I, while the increase in fluorescence (phase II) is caused only by EF–hand 4. The EF–hand 4 has lower affinity for Mg2+ compared to the other two EF–hands and causes only Ca2+–specific conformational change in GCAP–1. Mg2+–bound GCAP–1 is the RetGC activator form. EF–hands 2 and 3 are Ca2+/Mg2+–binding domains, and EF–hand 2 has the highest affinity for both Ca2+ and Mg2+. Under the conditions of light adaptation, when free Ca2+ drops to 20–25 nM, and free Mg2+ remains near 1 mM [2,3], EF–hand 2 has to be primarily occupied by Mg2+. The affinity of EF–hand 3 for the cations strongly depends on the presence of functional EF–hands 2 or 4, but in the wild type GCAP–1 its EF–hand 3 is also likely to be filled with Mg2+ in the light. In the dark, GCAP–1 becomes an inhibitor of RetGC, as a result of Mg2+ substitution for Ca2+. References.  Peshenko & Dizhoor (2004) J. Biol. Chem. 279, 16903–6;  Woodruff et al. (2002) J.Physiol. 542, 843–54;  Chen et al. (2003) J. Physiol. 553, 125–35.
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