Ryanodine receptors (RyRs) are intracellular, homotetrameric ion channels in muscle cells that gate the release of calcium (Ca 2+ ) from the sarcoplasmic reticulum (SR). RyR channels flood the myoplasm with Ca 2+ following depolarization of the muscle surface membrane, causing contraction. RyRs are regulated by myoplasmic Ca 2+ . Depolarization is accompanied by a modest influx of Ca 2+ through voltage-gated Ca 2+ channels, which activates RyR channels. Subsequently, RyRs are inactivated by the higher Ca 2+ levels present after SR Ca 2+ -release, thus creating a self-terminating burst of Ca 2+ during muscle contraction. C. elegans has a single RyR gene (
unc-68) that encodes a 5074 amino acid RyR protein. In body-wall muscle cells, UNC--68 channels are localized in SR vesicles between the surface membrane and the myofilament lattice. unc--68 null mutant animals are viable, but have defective locomotion. We are examining the biological role of Ca 2+ binding sites in UNC-68 RyRs that may be involved in activation or termination of Ca 2+ release in body-wall muscle cells. The predicted UNC-68 protein has two EF hand-type Ca 2+ -binding motifs at amino acids 4198-4246 (Sakube, et. al. 1997 JMB 267 :849). We have eliminated one or both EF hands using alanine substitution, and analyzed 45 Ca 2+ -binding to wild type and mutant fusion proteins containing the EF-hand region. Both the EF hand motifs bind 45 Ca 2+ , as shown by 45 Ca 2+ -overlays and equilibrium binding. We have expressed GFP-tagged
unc-68 genes containing wild-type EF-Hands or the EF-Hand mutations in unc--68 null mutant animals. Proper expression and localization of the GFP-tagged RyRs have been confirmed using in vivo localization of the GFP tag, and western blot analysis of purified, detergent-solubilized RyR proteins. Both intact EF hands are required for rescue of motility (i.e. only the WT gene rescues normal locomotion). The two genes with a single EF hand eliminated (EF1 + EF2 - or EF1 - EF2 + ), give partial rescue of motility, and restore sensitivity to paralysis by ryanodine. The gene lacking both EF-Hand calcium-binding sites does not rescue motility or restore sensitivity to ryanodine. Interestingly, when expressed in
unc-68(0)/+ or wild-type animals, arrays containing all three EF-Hand mutant RyR genes exert semi-dominant effects on motility. One interpretation of this semi-dominance is that the mutant RyR subunits form heterotetramers with Wild Type subunits. Further characterization of the purified mutant RyR channels should reveal whether the EF-Hand calcium-binding sites are involved in activation or inactivation of calcium release during body-wall muscle contraction.