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The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S+ rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+ phototransduction feedback and abnormally high free-Ca2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- mice lacking Ca2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca2+ levels in photoreceptors.SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase m

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.