title: The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.
abstract: Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca(2+) is a central step in the process of Ca(2+)-induced Ca(2+) release, but the molecular basis of RyR2 activation by cytosolic Ca(2+) is poorly defined. It has been proposed recently that the putative Ca(2+) binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca(2+) sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca(2+) activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca(2+)-dependent activation of [(3)H]ryanodine binding or the cytosolic Ca(2+) activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca(2+) activation of RyR2 and spontaneous Ca(2+) release in HEK293 cells during store Ca(2+) overload or store overload-induced Ca(2+) release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca(2+), it plays an important role in luminal Ca(2+) activation and SOICR.
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Wenting
authors.initials: W
authors.lastname: Guo
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Bo
authors.initials: B
authors.lastname: Sun
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Yundi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.affiliation: From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada swchen@ucalgary.ca.
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism*; D020832:EF Hand Motifs; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium
keywords: calcium; calcium channel; calcium imaging; calcium intracellular release; calcium transport; endoplasmic reticulum (ER); excitation-contraction coupling (E-C coupling); ryanodine receptor; sarcoplasmic reticulum; site-directed mutagenesis