THE ROLE OF CALCIUM-BINDING SITES IN S-MODULIN FUNCTION

S-modulin controls rhodopsin phosphorylation in a calcium-dependent ma nner, and it has been suggested that it modulates the light sensitivit y of the photoreceptor cell, S-modulin binds to the ROS membrane at hi gh Ca2+ concentration, and N-terminal myristoylation is necessary for this property (the calcium-myristoyl switch). S-modulin has four EF-ha nd motifs, of which two (EF-2 and -3) are functional. Here, we report on the roles of EF-2 and -3 in S-modulin function (calcium binding, me mbrane association, and inhibition of rhodopsin phosphorylation) by si te-directed mutants (E85M and E121M), Surprisingly, E121M, which has a mutation in EF-3, neither binds Ca2+ nor inhibits phosphorylation, In contrast, E85M binds one Ca2+ and has the same membrane affinity as w ild-type S-modulin, but has lost the ability to inhibit rhodopsin phos phorylation, It is suggested that the binding of Ca2+ to EF-3 is proba bly required for EF-2 to be a functional Ca2+-binding site and to indu ce exposure of the myristoyl group; and that the binding of Ca2+ to EF -2 is important for the interaction with rhodopsin kinase.