Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca 2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1)

Abstract

We explored the possibility that, in the regulation of an effector enzyme by a Ca2+-sensor protein, the actual Ca2+ sensitivity of the effector enzyme can be determined not only by the affinity of the Ca 2+-sensor protein for Ca2+ but also by the relative affinities of its Ca2+-bound versus Ca2+-free form for the effector enzyme. As a model, we used Ca2+-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg838Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca2+ sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca2+ range. We find that at physiological concentrations of Mg2+ this mutation increases the free Ca2+ concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 μM. Similar to rod outer segment cyclase, Ca 2+ sensitivity of recombinant RetGC-1 is strongly affected by Mg 2+, but the shift in Ca2+ sensitivity for the R838S mutant relative to the wild type is Mg2+-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca 2+-bound and Ca2+-free GCAP-1 and found that the net shift in Ca2+ sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca2+-free versus Ca2+-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca2+/GCAP is determined by both the affinity of GCAP for Ca2+ and relative affinities of the effector enzyme for the Ca2+-free versus Ca 2+-loaded GCAP.