Mg2+ Inhibits Cardiac SR Calcium Release and has Biphasic Effects on Calmodulin Binding to RyR2

Abstract

Mg2+ is a potent inhibitor of RyR channel gating, as demonstrated in lipid bilayer and SR vesicles. Less is known about Mg2+ effects on RyR2 function in the cardiac myocyte environment or how it might alter RyR2 interaction with accessory proteins like calmodulin (CaM), which by itself binds to, and inhibits RyR2 opening. We used permeabilized mouse ventricular cardiomyocytes to study the effects of Mg2+ on spontaneous Ca2+ release and CaM affinity for RyR2. We recorded Ca sparks at three [Mg2+]: 0.1 mM (low), 1 mM (physiological), and 3 mM (high). We found that in the presence of endogenous WT-CaM, with increasing [Mg2+], Ca spark frequency (CaSpF) decreased monotonically, and was accompanied by an increase in Ca spark amplitude and SR Ca2+ load. To test whether Mg2+ also modulates RyR2 interaction with accessory proteins, we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants (N54I and N98S). The apparent affinity of WT-CaM for RyR2 in myocytes was highest at 1 mM Mg2+ and decreased at both 0.1 mM and 3 mM Mg2+. The same pattern was found for N54I- and N98S-CaM. We conclude that in cardiomyocytes, Mg2+ decreases CaSpF and increases SR Ca2+ load. As [Mg2+] increases in the physiologically range (0.1 - 1 mM), part of the Ca spark inhibition may be due to enhanced CaM binding to RyR2. In contrast, pathophysiologically high [Mg2+] (3 mM) may (1) limit RyR2-CaM binding (and thus CaM inhibition of RyR2) and (2) still directly inhibit RyR2 opening.