Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) with high affinity, and it may act as a Ca2+-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca2+-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca2+-CaM, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca2+-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca2+-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca2+ release during oxidative stress.
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