Unveiling biochemical and physiological consequences of cone dystrophy-related mutations in GCAP1

Purpose Cone dystrophies are often associated with altered levels of calcium (Ca2+) and cyclic GMP (cGMP), the second messengers operating in the phototransduction cascade in rod and cone photoreceptors. By using a multiscale approach, we investigated the biochemical and physiological effects of four pathogenic point mutations identified in the guanylate cyclase-activating protein 1 (GCAP1), leading to the amino acid substitutions E89K, D100E, L151F and G159V. Methods Structure-function relationships were studied by biophysical methods, including circular dichroism to monitor secondary and tertiary structural changes in GCAP1 variants upon binding of Ca2+ and isothermal titration calorimetry to monitor the thermodynamics of Ca2+-binding. Experimental parameters describing the regulation of the target enzyme guanylate cyclase 1 (GC) by each GCAP1 variant were incorporated into a comprehensive kinetic model of phototransduction, in order to assess the effect of each individual point mutation on the whole cell response. Results Wild type and cone dystrophy-related point mutations in GCAP1 showed large differences in Ca2+-binding and GC regulation but, except for E89K, the structural effects of all the tested mutations are minor and involve mostly a slight rearrangement of aromatic residues in the Ca2+-bound form. System-level modeling suggests that the main effect of all point mutations on the photoresponse kinetics is a perturbation of the photocurrent shape consisting in increased amplitude and prolonged duration. However, the effect is strongly dependent on the expression levels of pathogenic GCAP1 forms as compared to the wild-type form. Conclusion Our data suggest that a multiscale approach combining biochemistry, biophysics and systems biology strategies allows a deep molecular understanding of dysfunctional states in photoreceptors in cone-dystrophy conditions. In particular, we conclude that the contribution of GCAP1 to the dynamic synthesis of cGMP in rod cells depends on the expression level of the wild type form, and in the case of high expression levels of cone-dystrophy GCAP1 mutants it would not contribute at all to shaping the cGMP rate, which becomes dynamically regulated solely by the other present Ca2+-sensor GCAP2.