Differences in the regulation of RyR2 from human, sheep, and rat by Ca2+ and Mg2+ in the cytoplasm and in the lumen of the sarcoplasmic reticulum

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca ²⁺ and Mg ²⁺ plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca ²⁺-and Mg ²⁺-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca ²⁺ activation (K _a = 4 μM) and inhibition by cytoplasmic Mg ²⁺ (Ki = 10 μM at 100 nM Ca ²⁺) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca ²⁺, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg ²⁺ inhibition than those from sheep and rat. The K _a values for luminal Ca ²⁺ activation were similar in the three species (35 μM for human, 12 μM for sheep, and 10 μM for rat). From the relationship between open probability and luminal [Ca ²⁺], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ~10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg ²⁺ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca ²⁺ and Mg ²⁺ only occurred when cytoplasmic [Ca ²⁺] was <3 μM. The activation response of RyR2 to luminal and cytoplasmic Ca ²⁺ was strongly dependent on the Mg ²⁺ concentration. Addition of physiological levels (1 mM) of Mg ²⁺ raised the K _a for cytoplasmic Ca ²⁺ to 30 μM (human and sheep) or 90 μM (rat) and raised the K _a for luminal Ca ²⁺ to ~1 mM in all species. This is the first report of the regulation by Ca ²⁺ and Mg ²⁺ of native RyR2 receptor activity from healthy human hearts.