Regulation Of Photoreceptor Guanylyl Cyclase By Ca2+/Mg2+ Exchange In GCAPs

Abstract

Photon absorption by rods and cones activates transduction cascade that shuts down cGMP-gated channels and thus decreases free Ca2+ concentrations in outer segment. The Ca2+ feedback, which activates guanylyl cyclase (retGC) through guanylyl cyclase activating proteins (GCAPs), accelerates cGMP re-synthesis in photoreceptors and thus expedites their recovery. GCAP1 and GCAP2, two ubiquitous among vertebrate species GCAPs that sequentially activate retGC during physiological response of rods to light, are Ca2+/Mg2+-binding proteins. They have one non-metal binding EF-hand, EF1, and three metal-binding EF-hands - EF2, EF3, and EF4. For each metal-binding EF-hand in GCAP1 we found point mutations that can block binding of Ca2+, but not Mg2+, and those that can block both Ca2+ and Mg2+ binding. We tested their effects on activation of retGC at physiological Mg2+ and either low Ca2+ (conditions representing light adaptation) or high Ca2+ (dark adaptation). Mg2+ binding in EF-2 and EF3 was essential for activation of retGC in the conditions of light adaptation. Mg2+ in EF2 was especially critical for the binding of GCAP1-GFP to retGC1 in co-transfected HEK293 cells, as revealed by confocal fluorescence microscopy. Mg2+ binding in EF4 contributed to neither retGC1 docking nor its activation. Instead, the replacement of Mg2+ by Ca2+ in this domain in the conditions of dark adaptation was the key event that switched the cyclase off. The Mg2+/Ca2+exchange in EF3 was required for the subsequent binding of Ca2+ in EF4. Contrary to EF3 and EF4, Mg2+/Ca2+ exchange in EF2 was not essential for retGC inhibition. Binding of Mg2+ versus Ca2+ causes characteristic changes in the intrinsic Trp fluorescence of GCAP1 corresponding to its activator versus inhibitor states, including the non-metal binding EF1.