Identification of Ca2+– and Mg2+– Specific EF–Hands in GCAP–1

Abstract

Abstract: : 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⁺ [1]. 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. [1] Peshenko & Dizhoor (2004) J. Biol. Chem. 279, 16903–6; [2] Woodruff et al. (2002) J.Physiol. 542, 843–54; [3] Chen et al. (2003) J. Physiol. 553, 125–35.

Keywords: signal transduction • calcium • photoreceptors