Characterization of the Calcium-Binding and Peptide-Binding Properties of Arrhythmogenic Calmodulin Mutants

Abstract

Calmodulin (CaM) is a ubiquitous calcium-binding protein that is integral to a variety of cellular functions including memory, immune response, and cardiac muscle contraction. Although CaM has no intrinsic enzyme activity, it binds and subsequently regulates hundreds of enzymes and ion channels in response to changes in cytosolic Ca2+ concentration. Missense mutations in CaM have recently been identified in individuals with symptoms of catecholaminageric polymorphic ventricular tachycardia (CPVT), a type of cardiac arrhythmia. The mechanisms behind the effects of these mutations remain unknown. We have purified and characterized the CaM mutants N54I and N98S in terms of their Ca2+ affinity and kinetics, as well as their interaction with the CaM-binding domain of the cardiac ryanodine receptor (RYR2).In agreement with previous studies, the steady-state Ca2+ sensitivity for CaM N54I was not different from WT CaM, while CaM N98S had decreased C-terminal Ca2+ sensitivity. Consistent with the C-terminal Ca2+ sensitivities, the C-terminal Ca2+ dissociation rate for N54I was not different from WT CaM, while N98S had an order of magnitude faster Ca2+ dissociation rate. In the presence of the RYR2 peptide, the C-terminal Ca2+ dissociation rate for N98S was two orders of magnitude faster than WT, while that of N53I was not changed. However, in the presence of the RYR2 peptide, both N-terminal Ca2+ dissociation rates were two-fold faster than WT. Thus a defective N-terminal CaM-RYR2 interaction may be the root cause of CPVT induced by these mutations. These studies will help to elucidate the mechanism by which CaM mutations cause cardiac arrhythmias, paving the way for the development of therapeutic calmodulins to treat cardiac disease.