Lead is a highly toxic metal that severely perturbs physiological processes even at sub-micromolar levels, often by disrupting the Ca2+ signaling pathways. Recently, Pb2+-associated cardiac toxicity has emerged, with potential involvement of both the ubiquitous Ca2+ sensor protein calmodulin (CaM) and ryanodine receptors. In this work, we explored the hypothesis that Pb2+ contributes to the pathological phenotype of CaM variants associated with congenital arrhythmias. We performed a thorough spectroscopic and computational characterization of CaM conformational switches in the co-presence of Pb2+ and four missense mutations associated with congenital arrhythmias, namely N53I, N97S, E104A and F141L, and analyzed their effects on the recognition of a target peptide of RyR2. When bound to any of the CaM variants, Pb2+ is difficult to displace even under equimolar Ca2+ concentrations, thus locking all CaM variants in a specific conformation, which exhibits characteristics of coiled-coil assemblies. All arrhythmia-associated variants appear to be more susceptible to Pb2+ than WT CaM, as the conformational transition towards the coiled-coil conformation occurs at lower Pb2+, regardless of the presence of Ca2+, with altered cooperativity. The presence of arrhythmia-associated mutations specifically alters the cation coordination of CaM variants, in some cases involving allosteric communication between the EF-hands in the two domains. Finally, while wild type CaM increases the affinity for the RyR2 target in the presence of Pb2+, no specific pattern could be detected for all other variants, ruling out a synergistic effect of Pb2+ and mutations in the recognition process.
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