Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket

Arrhythmia-Associated Calmodulin E105A Mutation Alters the Binding Affinity of CaM to a Ryanodine Receptor 2 CaM-Binding Pocket

“…Many studies have reported different RyR2 regions as potential CaM-binding sequences, with the sequence that lies within the residues 3583-3603aa of RyR2 as the most well-established RyR2 CaM-binding region [16,[39][40][41][42]. We recently showed that CaM interacts with two peptides that correspond to two different human RyR2 CaM-binding regions (3584-3602aa and 4255-4271aa), and we proposed that these two regions might contribute to a putative intra-subunit CaM-binding pocket [38].…”

“…To gain further insight into how the CaM E105A mutation leads to severe cardiac arrhythmia and determine quantitatively how this specific mutation alters the association of CaM with RyR2 leading to diminished inhibition, we initially bacterially expressed and purified large quantities of recombinant CaM WT and CaM E105A proteins, as we have previously described [37]. We then performed ITC experiments to investigate and compare the interactions of CaM WT and CaM E105A mutant protein with two synthetic peptides that correspond to the two human RyR2 regions, which we have recently proposed to contribute to a putative intra-subunit CaM-binding pocket [38]. The first peptide (B) corresponds to the well-established RyR2 CaM-binding region (3581-3607aa), while the second peptide (F) corresponds to another putative CaM-binding region (4240-4277aa), which we previously found to interact with significant affinity with CaM, both in the presence and absence of Ca 2+ [38].…”

“…We then performed ITC experiments to investigate and compare the interactions of CaM WT and CaM E105A mutant protein with two synthetic peptides that correspond to the two human RyR2 regions, which we have recently proposed to contribute to a putative intra-subunit CaM-binding pocket [38]. The first peptide (B) corresponds to the well-established RyR2 CaM-binding region (3581-3607aa), while the second peptide (F) corresponds to another putative CaM-binding region (4240-4277aa), which we previously found to interact with significant affinity with CaM, both in the presence and absence of Ca 2+ [38]. Figure 1 shows typical ITC profiles for the interactions between peptide B and CaM WT and CaM E105 proteins in holo-buffer, at 25 • C. The reactions of CaM WT and CaM E105 proteins with peptide B appeared to be exothermic, with the large exothermic signals signifying the formation of a network of favorable bonds between the receptor and ligand.…”

“…However, different molecular mechanisms have been proposed through which CaM missense mutations can lead to the aforementioned arrhythmogenic phenotypes. One primary mechanism involves abnormal cytosolic Ca 2+ levels produced as a result of increased Ca 2+ "leakage" from SR due to reduced/abolished inhibition of RyR2 [29][30][31][32][33][34][35][36][37][38]. Many studies have reported different RyR2 regions as potential CaM-binding sequences, with the sequence that lies within the residues 3583-3603aa of RyR2 as the most well-established RyR2 CaM-binding region [16,[39][40][41][42].…”

“…We have previously characterized a de novo LQTS-associated missense CaM mutation (E105A) which was identified in a 6-year-old boy, who experienced an aborted first episode of cardiac arrest [27,37]. In that study, we proposed that CaM E105A mutation can dysregulate normal cardiac function in zebrafish by a complex mechanism that involves alterations in both CaM-Ca 2+ and CaM-RyR2 interactions [38].…”