Molecular basis of Ca2+ activation of the mouse cardiac Ca2+ release channel (ryanodine receptor)

Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little i s known about the molecular basis of activation of RyR2 by Ca2+. In this st udy, we investigated the role in Ca2+ sensing of the conserved glutamate 39 87 located in the predicted transmembrane segment M2 of the mouse RyR2. Sin gle point initiation of this conserved glutamate to alanine (E3987A) reduce d markedly the sensitivty of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [H-3] r yanodine binding assay. However, this mutation did not alter the affinity o f [H-3]ryanodine binding and the single-channel conductance. In addition, t he E3987A mutant channel was activated by caffeine and ATP. was inhibited b y Mg2+, and was modified by ryanodine in a fashion similar to that of the w ild-type channel. Coexpression of the wild-type and Mutant E3987A RyR2 prot eins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the vie w, that glutamate 3987 is a major determinant of Ca2+ sensitivity to activa tion of the Mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results Of this Study also provide initial insights into the structural and functional pro perties of the mouse RyR2, which should be useful for studying RyR2 functio n and regulation in genetically modified mouse models.