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The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca 2+ concentrations decline, and decelerates the cyclase in the dark, when Ca 2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 ( GUCA1A ) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca 2+ -dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca 2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca 2+ concentrations characteristic of dark-adapted photoreceptors. Ca 2+ -dependent increase in Trp 94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca 2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca 2+ , and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca 2+ sensitivity of the cyclase within the physiological range of intracellular Ca 2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly 86 , leading to abnormally high affinity for the target enzyme and reduced Ca 2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca 2+ influx in photoreceptors in the dark., (© 2019 Peshenko et al.)