Abstract: Phototransduction in vertebrate photoreceptor cells is controlled by Ca2+-dependent feedback loops involving the membrane-bound guanylate cyclase GC-E that synthesizes the second messenger guanosine-3′,5′-cyclic monophosphate. Intra-cellular Ca2+-sensor proteins named guanylate cyclase-activating proteins (GCAPs) regulate the activity of GC-E by switching from a Ca2+-bound inhibiting state to a Ca2+-free/Mg2+-bound activating state. The gene GUCY2D encodes for human GC-E, and mutations in GUCY2D are often associated with an imbalance of Ca2+ and cGMP homeostasis causing retinal disorders. Here, we investigate the Ca2+-dependent inhibition of the constitutively active GC-E mutant V902L. The inhibition is not mediated by GCAP variants but by Ca2+ replacing Mg2+ in the catalytic center. Distant from the cyclase catalytic domain is an α-helical domain containing a highly conserved helix-turn-helix motif. Mutating the critical amino acid position 804 from leucine to proline left the principal activation mechanism intact but resulted in a lower level of catalytic efficiency. Our experimental analysis of amino acid positions in two distant GC-E domains implied an allosteric communication pathway connecting the α-helical and the cyclase catalytic domains. A computational connectivity analysis unveiled critical differences between wildtype GC-E and the mutant V902L in the allosteric network of critical amino acid positions. URL