We recently identified the first two human missense mutations in a calmodulin (CaM) gene (CALM1) and linked these to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death in young individuals1. More CaM mutations have since been identified in CALM1 and also in the other two CaM genes (CALM2 and CALM3). All CaM mutations are associated with severe ventricular arrhythmias. CaM regulates several key proteins governing cardiac excitation-contraction coupling (ECC), including the cardiac ryanodine receptor (RyR2) Ca2+ release channel. RyR2 mutations also dominantly cause CPVT, where the mutations increase the channel sensitivity to activation and enhance the propensity for pro-arrhythmogenic spontaneous Ca2+ release. Here we investigated the effect of CPVT-linked CaM mutations (N53I and N97S) and two CaM mutations identified in individuals with early onset severe long QT syndrome (LQTS) (D95V and D129G)2, on spontaneous Ca2+ release in HEK293 cells expressing the RyR2 channel. Furthermore, we studied the impact of these mutations on the interactions between CaM and a peptide corresponding to the RyR2 CaM binding domain (CaMBD) residue number 3581-3611, and the Ca2+ dependence of this interaction. In contrast to WT CaM, all four CaM mutations slightly, but significantly, lowered the activation threshold at which spontaneous Ca2+ release occurs. In addition, all CaM mutations significantly reduced the threshold at which spontaneous Ca2+ release terminates. Taken together, all CaM mutations induced an excessive fractional Ca2+ release from internal Ca2+ stores. Interestingly, we demonstrate that the presence of the RyR2 CaMBD decrease the WT CaM C-lobe apparent KD for Ca2+ binding >80 fold to 30 nM. This suggests that Ca2+ binding to the C-lobe of CaM would be nearly saturated at diastolic Ca2+ concentrations (~100 nM), and that the C-lobe of CaM would constitutively anchor to RyR2 in a Ca2+ bound state throughout the ECC cycle. Conversely, the N-lobe of CaM seems poised to be sensing changes in physiological Ca2+ with an apparent KD of ~800 nM in the presence of RyR2 CaMBD. The D95V, N97S and D129G mutations lowered the affinity of Ca2+ binding of the C-lobe of CaM, to apparent KDs of ~ 140, 150, and 4000 nM, respectively, consistent with the critical role of these residues in Ca2+ binding to the C-lobe. Thus, we suggest that these mutations may shift to an apo-CaM binding state during diastole, leading to dysregulation of RyR2 mediated Ca2+ release. Despite the pronounced impact on RyR2 mediated Ca2+ release, the N-lobe N53I mutation only imposed a small lowering of the N-lobe Ca2+ affinity (KD ~1200 nM). Thus, the RyR2 mediated Ca2+ release is either highly sensitive to minor changes in CaM N-lobe Ca2+ affinity, or the N53I mutation perturbs interactions between CaM and RyR2 outside the 3587-3605 CaM binding domain. We conclude that dysregulation of RyR2 mediated SR calcium release is likely a major molecular disease determinant of CPVT-linked CaM mutations, and an important component of the clinical manifestation for LQTS-causing CaM mutations.
|Publication date||1 Jul 2015|
|Number of pages||1|
|Publication status||Published - 1 Jul 2015|
|Event||19th International Symposium on Calcium Binding Proteins and Calcium Function In Health and Disease - Vanderbilt University, Nashville, United States|
Duration: 30 May 2015 → 4 Jun 2015
|Conference||19th International Symposium on Calcium Binding Proteins and Calcium Function In Health and Disease|
|Period||30/05/2015 → 04/06/2015|