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| - Factors that Determine Ca2+ Sensitivity of Photoreceptor Guanylyl Cyclase. KineticAnalysis of the Interaction between the Ca2+-Bound and the Ca2+-Free GuanylylCyclase Activating Proteins (GCAPs) and Recombinant Photoreceptor GuanylylCyclase 1 (RetGC-1)
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| - We explored the possibility that, in the regulation of an effector enzyme by a Ca2+-sensorprotein, the actual Ca2+ sensitivity of the effector enzyme can be determined not only by the affinity ofthe Ca2+-sensor protein for Ca2+ but also by the relative affinities of its Ca2+-bound versus Ca2+-freeform for the effector enzyme. As a model, we used Ca2+-sensitive activation of photoreceptor guanylylcyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg838Ser in RetGC-1found in human patients with cone−rod dystrophy is known to shift the Ca2+ sensitivity of RetGC-1regulation by GCAP-1 to a higher Ca2+ range. We find that at physiological concentrations of Mg2+ thismutation increases the free Ca2+ concentration required for half-maximal inhibition of the cyclase from0.27 to 0.61 μM. Similar to rod outer segment cyclase, Ca2+ sensitivity of recombinant RetGC-1 is stronglyaffected by Mg2+, but the shift in Ca2+ sensitivity for the R838S mutant relative to the wild type isMg2+-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 forboth Ca2+-bound and Ca2+-free GCAP-1 and found that the net shift in Ca2+ sensitivity of the R838SRetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclasefor the Ca2+-free versus Ca2+-loaded GCAP-1. Our findings confirm that the dynamic range for RetGCregulation by Ca2+/GCAP is determined by both the affinity of GCAP for Ca2+ and relative affinities ofthe effector enzyme for the Ca2+-free versus Ca2+-loaded GCAP.
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