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J. Biol. Chem., Vol. 281, Issue 33, 23830-23841, August 18, 2006

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Ca²⁺ and Mg²⁺ Binding Properties of GCAP-1

EVIDENCE THAT Mg²⁺-BOUND FORM IS THE PHYSIOLOGICAL ACTIVATOR OF PHOTORECEPTOR GUANYLYL CYCLASE^*

From the Hafter Research Laboratories, Pennsylvania College of Optometry, Elkins Park, Pennsylvania 19027

Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca²⁺ in the light and inhibits it in the dark when Ca²⁺ concentrations rise. We present the first direct evidence that Mg²⁺-bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP-1, three bound Ca²⁺ ions and could exchange Ca²⁺ for Mg²⁺. At concentrations of free Ca²⁺ and Mg²⁺ typical for the light-adapted photoreceptors, all three metal-binding EF-hands were predominantly occupied by Mg², and the presence of bound Mg²⁺ in GCAP-1 was essential for its ability to stimulate RetGC-1. In the Mg²⁺-bound form of GCAP-1 all three Trp residues became more exposed to the polar environment compared with its apo form. The replacement of Mg²⁺ by Ca²⁺ in the EF-hands 2 and 3 further exposed Trp-21 to the solution in a non-metal-binding EF-hand domain 1 that interacts with RetGC. Contrary to that, replacement of Mg²⁺ by Ca²⁺ in the EF-hand 4 moved Trp-94 in the entering -helix of the EF-hand 3 back to the non-polar environment. Our results demonstrate that Mg²⁺ regulates GCAP-1 not only by adjusting its Ca²⁺ sensitivity to the physiological conditions in photoreceptors but also by creating the conformation required for RetGC stimulation.

Received for publication, January 10, 2006 , and in revised form, June 20, 2006.

^* This work was supported by National Institutes of Health Grant EY11522 and by Lions of Pennsylvania Sight Conservation Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Table 1.

¹ The Martin and Florence Hafter Professor of Pharmacology. To whom correspondence should be addressed: Pennsylvania College of Optometry, 8360 Old York Rd., Elkins Park, PA 19027. Tel.: 215-780-1468; Fax: 215-780-1464; E-mail: adizhoor{at}pco.edu.

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