Studies were initiated to define the equilibria of calmodulin binding to the skeletal muscle sarcoplasmic reticulum (SR) Ca2+-release channel protein in native SR vesicles. Calmodulin affinity-labeling experiments indicated that the major calmodulin receptor in heavy SR preparations was a protein of M(r) > 450000, corresponding to the Ca2+-release channel protein. [H-3]Ryanodine-binding assays indicated 10.6 +/- 0.9 pmol of high-affinity ryanodine binding per milligram of SR protein. Wheat germ calmodulin was derivatized with rhodamine-x-maleimide. The affinity and binding capacity of the channel protein in SR vesicles for the derivatized calmodulin (Rh-CaM) were determined by fluorescence anisotropy in the presence of (1) 1 mM EGTA, (2) 0.1 mM CaCl2, and (3) 0.1 mM CaCl2 plus 1 mM MgCl2. In the presence of EGTA, Rh-CaM bound to the channel protein with a K(d) of 8.6 +/- 0.8 nM and a B(max) of 229 +/- 7 pmol/mg, suggesting that calmodulin binds to the channel protein at [Ca2+] comparable to that in resting muscle. In the presence of 0.1 mM CaCl2, the binding equilibrium shifted to a two-site ligand-binding model; the high-affinity class of sites had a B(max1) of 54 +/- 7 pmol/mg and a Dd1 of 4.3 +/- 1.1 nM, while the lower affinity class of sites had a B(max2) of 166 +/- 28 pmol/mg and a K(d2) of 239 +/- 102 nM. In the presence of both Ca2+ and Mg2+, there was a further change in the Rh-CaM/channel protein interaction. B(max1) was 10.0 +/- 0.8 pmol/mg, and K(d1) was 0.10 +/- 0.03 nM; B(max2) was 70 +/- 2 pmol/mg, and K(d2) was 17 +/- 1 nM. These data are consistent with the hypothesis that there are multiple calmodulin-binding sites on each channel protein subunit with the affinities of these calmodulin-binding sites depending on the concentrations of Ca2+ and Mg2+. Thus, the binding of calmodulin to the SR Ca2+ channel is regulated by modulators of the Ca2+ channel activity itself, and this novel regulation is likely to be important in the mechanism of excitation-contraction.
