Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors

Abstract

Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca 2+-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca2+ and inhibit RetGC in a Ca 2+-sensitive manner. The reported data for the RetGC inhibition by Ca2+/GCAPs in vitro are in disagreement with the free Ca 2+ levels found in mammalian photoreceptors (Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843-854). We have found that binding of Mg 2+ dramatically affects both Ca2+-dependent conformational changes in GCAP-1 and Ca2+ sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg2+ concentrations ([Mg]f) from 5.0 mM to 0.5 mM decreases the free Ca2+ concentration required for half-maximal inhibition of RetGC ([Ca] 1/2) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 μM to 0.16 and 0.03 μM, respectively. A similar effect of Mg2+ on Ca 2+ sensitivity of RetGC by endogenous GCAPs was observed in mouse retina. Analysis of the [Ca]1/2 changes as a function of [Mg] f in mouse retina shows that the [Ca]1/2 becomes consistent with the range of 23-250 nM free Ca2+ found in mouse photoreceptors only if the [Mg]f in the photoreceptors is near 1 mM. Our data demonstrate that GCAPs are Ca2+/Mg2+ sensor proteins. While Ca2+ binding is essential for cyclase activation and inhibition, Mg2+ binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca2+.