Worm Breeder's Gazette 12(4): 69 (October 1, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Characterization of the C. elegans Ryanodine Receptor

Ed Maryon[1], YoungKee Kim[2], Roberto Coronado[2], Phil Anderson[1]

[1]Depts of Genetics, University of Wisconsin, Madison WI 53706
[2]Depts of Physiology, University of Wisconsin, Madison WI 53706

The vertebrate sarcoplasmic reticulum contains ion channels responsible for the rapid, massive release of calcium into the cytoplasm during muscle contraction. These high-conductance calcium release channels are known as ryanodine receptors. Ryanodine is a plant alkaloid that binds to open channels, locking them in an open state and causing contractile paralysis of muscle fibers. Distinct isoforms of ryanodine receptors are found in skeletal or cardiac muscle in mammals, encoded by separate genes. Both of these genes encode large proteins of about 5000 amino acids (565 kd). Functional ryanodine receptors are homotetramers.

We and others have made several observations that suggest that ryanodine receptors exist in nematodes. Treatment of wild-type C. elegans with ryanodine results in hypercontractive paralysis, suggesting an effect of the drug on muscle. Purified C. elegans membranes contain a high affinity ryanodine binding activity. Sucrose gradient fractions with the greatest binding activity also contain high conductance ion channels that are gated by ryanodine (Kim et al. 1992 Biophysical J. in press). In the last WBG, (Vol. 12 #3 p. 51), Sakube, Ando and Kagawa reported the identification of a cDNA clone homologous to the C-terminus of mammalian ryanodine receptors. We obtained a 1.2 kb clone ( cm16 c2 )from the sequencing consortium that is homologous to the mammalian cardiac ryanodine receptor (WBG 12 #2, p26 ).This clone hybridized to two overlapping YACs on LG V near mec-l. The hybridization patterns of both cm16 c2 and the cDNA clone isolated by Sakube, et al. suggest that they are from the same gene (H. Kagawa, personal communication). We sequenced cm16 c2 and found an ORF that was =30% identical to various mammalian ryanodine receptors between aa residues 3300-3700. When used as a probe on northern blots, cm16 c2 hybridizes to a very large polyA+ RNA (> 10 kb; mammalian ryanodine receptor mRNAs are> 15 kb). Southern hybridization indicates that the cm16 c2 sequence is single-copy.

We are attempting to use the paralysis of wild type animals by ryanodine as a selection for mutations in the ryanodine receptor gene. 105 EMS-treated haploid genomes were screened for mutations enabling animals to thrash in liquid containing paralytic concentrations of ryanodine (rya (r)). The most frequently isolated rya(r) mutants were alleles of unc-22 ,as shown by their failure to complement the twitching phenotype of e66 .In addition to the apparent alleles of unc-22 ,12 other mutants were isolated having varying degrees of resistance to ryanodine. These mutants comprise at least 6 complementation groups. Some of these have been mapped to LG I, III, and IV. 11 of the mutants are recessive, the other is semi-dominant. Certain mutants also exhibit visible phenotypes (t.s. growth, Unc, Egl, semi-Dpy, etc.), but none so far have mapped to LG V. Using a PCR screen of deficiency homozygote embryos, cm16 c2 was placed on the genetic map between the right-hand breakpoints of sDf20 and nDf18 .We are screening for rya (r) mutants on LG V using non-complementation of sDf20 .We hope to find mutants in the C. elegans ryanodine receptor with altered gating properties, and to characterize these mutants at the molecular level.

Literature Cited:

Kim et al. 1992 Biophysical J. in press.

WBG, (Vol. 12 #3 p. 51).

WBG 12 #2, p26 .

H. Kagawa, personal communication.