We next assayed the degree to which mutant GCAP1-I143NT stimulated GC as a function of [Ca
2+]. We expected incomplete inactivation of GC1 at the high Ca
2+ levels found in dark-adapted photoreceptors, similar to what has been reported for GCAP1-Y99C
22 23 and GCAP1-E155G.
9 As shown in
Figure 2B , GCAP1-I143NT was active at low Ca
2+ (<200 nM), as was wild-type GCAP1, but the mutant remained active (30%–50%) even at high dark [Ca
2+]
free-levels (∼700 nM) when wild-type GCAP1 was inhibited. Similar incomplete suppression of GC activity was previously found with a GCAP1-Y99C mutation affecting EF3.
23 The mutant GCAP1-E155G exhibited similar incomplete suppression of GC activity, suggesting that in both mutants, as evidenced by the rightward shift in the Ca
2+-titration curve, EF4 binds Ca
2+ with an impaired affinity. This conclusion is consistent with our previous finding that the GCAP1-E155D
36 mutant, which is not able to bind Ca
2+ to this loop, showed activity even at high, nonphysiological Ca
2+ levels (50 μM [Ca
2+]
free). Mutagenesis experiments showed that when both EF3 and EF4 of GCAP1 are defective and allow no Ca
2+ binding, GCAP1 was converted into a Ca
2+-insensitive GC stimulator (Fig. 6B in Ref.
36 ). Taken together, the findings indicate that Ca
2+ binding by EF3 and EF4 is critical for inactivation of GCAP1, and dissociation of Ca
2+ from these sites converts GCAP1 from the inactive to an active state. Mutations affecting only one of these sites will partially impair GC inhibition, leading to persistent stimulation in dark-adapted photoreceptors.