Guanylyl cyclase-activating proteins (GCAPs) are Ca2+/Mg2+ sensors: implications for photoreceptor guanylyl cyclase (RetGC) regulation in mammalian photoreceptors.
Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca(2+)-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca(2+) and inhibit RetGC in a Ca(2+)-sensitive manner. The reported data for the RetGC inhibition by Ca(2+)/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 Ca(2+)-dependent conformational changes in GCAP-1 and Ca(2+) sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg(2+) concentrations ([Mg](f)) from 5.0 mm to 0.5 mm decreases the free Ca(2+) concentration required for half-maximal inhibition of RetGC ([Ca]((1/2))) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 microm to 0.16 and 0.03 microm, respectively. A similar effect of Mg(2+) 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 Ca(2+) found in mouse photoreceptors only if the [Mg](f) in the photoreceptors is near 1 mm. Our data demonstrate that GCAPs are Ca(2+)/Mg(2+) sensor proteins. While Ca(2+) binding is essential for cyclase activation and inhibition, Mg(2+) binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca(2+).
Hafter Research Laboratory, Pennsylvania College of Optometry, Elkins Park, Pennsylvania 19027.
Igor V
IV
Peshenko
Alexander M
AM
Dizhoor
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins; D008027:Light; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry; D012160:Retina / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; C496737:Guca1a protein, mouse; C496740:Guca1b protein, mouse; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C110715:guanylate cyclase 1; D014364:Tryptophan; D006162:Guanylate Cyclase; D008274:Magnesium; D002118:Calcium