Oxidation of Met144 and Met145 in Calmodulin Blocks Calmodulin Dependent Activation of the Plasma Membrane Ca-ATPase

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Abstract
Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met144 and Met145 near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met144 and Met145. Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met144 and Met145 or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50−60% reduction in the level of enzyme activation. Oxidation of Met144 is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met144 and Met145 in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.