To characterize excitation-contraction coupling in C. elegans, we have applied two approaches. First, we isolated a mutant having abnormal response against ketamine, an anesthetic in vertebrates.
unc-68(
kh30), the novel semi-dominant allele of
unc-68 mutation that was initially isolated as
kra-1(
kh30), exhibited strict ketamine-dependent convulsions followed by paralysis (7th C. elegans meeting, 1989). Second, we cloned and analyzed the C. elegans ryanodine receptor gene,
ryr-1. CeRYR had a homology to those of Drosophila and the cardiac type of rabbit at the rate of 47% and 42% respectively. Ryr-1 promoter/lacZ plasmids were expressed in body wall and pharyngeal muscles, were also expressed in neurons and intestinal cells (10th meeting, 1995). We identified the mutation sites of
kra-1 and
unc-68 in the
ryr-1 gene (WC Meeting, 1996). The
kra-1(
kh30) mutation was a Ser1,444Asn substitution at a putative PKC phosphorylation site of CeRYR. The
unc-68(
e540) mutation was in a splice acceptor site caused a stop codon at the middle in the
ryr-1 gene. The
kh30 mutation locates in the most hydrophilic region of the foot structure. This mutation region may be responsible for the specific modification or interaction with other regulatory molecules. We also confirmed that
unc-68 mutation was a defect of the
ryr-1 gene. pCRYR1, the designed cosmid vector including the entire
ryr-1 coding sequence and the essential 5'-regulatory sequences, rescued mutation phenotypes of the
unc-68(
e540) animal. Injected
e540 animals segregated both Rol (non-Unc) and the wild type progenies.
e540::ExpCRYR1 animals moved at 73% of the wild type, and recovered the sensitivity to 1 mM ryanodine. In contrast,
e540 and
r1158, a Tc1 induced deletion allele of
ryr-1 showed only 20-23% motility of the wild type and lacked ryanodine sensitivity (Lewis et al. 1980, Maryon & Anderson, 10th meeting, 1995). The reason why it took so long to get the final result was follows; 1) poor information about the gap between the genetic and the physical map at that time, 2) the
unc-68 mutation has a weak phenotype, 3)
unc-68 is a huge gene that spans more than 30 kb and encodes a predicted polypeptide of 5,071 amino acid residues. Detailed analysis on the
kh30 animal is useful to know the calcium signaling pathway from the voltage sensitive calcium channel to the muscle fibers.