Role of calmodulin methionine residues in mediating productive association with cardiac ryanodine receptors

Abstract

Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity

“…This suggests that both CaM and RyR2 oxidation contribute to the reduced CaM binding. It also suggests that CaM was not fully oxidized, by our in vitro pre-exposure to 50 μM H 2 O 2 (Balog used 1000 times higher H 2 O 2 concentration for 24 hr; [37]).…”

Oxidation of RyR2 Has a Biphasic Effect on the Threshold for Store Overload-Induced Calcium Release

“…Although direct regulation of RyR2 by methionine oxidation has not been reported, calmodulin (CaM) a protein that associates and regulates RyR2 is known to undergo methionine oxidation (31). The oxidation of CaM reduces its affinity for RyR2 leading to dissociation, destabilization of RyR2, and a subsequent increase in RyR2 activity (32). HEK293 endogenously express CaM (33), which would likely be dissociated by the high concentrations of H 2 O 2 in our assays.…”

Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II

“…The effect of oxidation on calcium/CaM signaling has previously been characterized for the interaction of CaM with a number of signaling partners, including PMCA, MLCK and RyR (Balog et al, 2003(Balog et al, , 2006Bartlett et al, 2003;Boschek et al, 2008;Chen et al, 2005;Gao et al, 1998aGao et al, , 2001Jas and Kuczera, 2002;Osborn et al, 2004;Robison et al, 2007;Slaughter et al, 2007;Yao et al, 1996;Yin et al, 2000). In these studies it was found that site specific oxidation at critical methionine residues (Met144/145 for PMCA or Met124 for RyR and MLCK) could account for the majority of the effect of oxidation on calcium/CaM signaling.…”

“…Furthermore, previous reports have shown that global methionine oxidation in CaM decreases its affinity for calcium, and interferes with binding and activation of a number of signaling partners (Schallreuter et al, 2007;Slaughter et al, 2005). These include the plasma membrane calcium ATPase (PMCA), myosin light chain kinase (MLCK), ryanodine receptor (RyR) and also CaMKII (Balog et al, 2003;Balog et al, 2006;Bartlett et al, 2003;Boschek et al, 2008;Chen et al, 2005;Gao et al, 1998aGao et al, , 2001Jas and Kuczera, 2002;Osborn et al, 2004;Robison et al, 2007;Slaughter et al, 2007;Yao et al, 1996;Yin et al, 2000). When CaM becomes progressively more oxidized, it shows reduced ability to induce autophosphorylation, substrate phosphorylation and substrate binding behavior in CaMKII, and no longer prevents the inhibitory autophosphorylation.…”