Rare altered-function mutations in the genes
unc-93,
sup-9, or
sup-10 result in the abnormal regulation or coordination of muscle contraction. These mutants move sluggishly, are unable to lay eggs, and exhibit a rubber-band phenotype: when worms are prodded on the head, they contract and relax along the entire length of the body without moving backwards1,2,3. Loss-of-function mutations in any of these three genes completely suppress any rubber-band mutation, suggesting that all three genes act at the same step, possibly by encoding subunits of a protein complex. Loss-of-function mutations in a fourth gene,
sup-18, completely suppress the rubber-band phenotype caused by
sup-10(
n983) and partially suppress the
unc-93(
e1500) and
sup-9(
n1550) rubber-band alleles2.
unc-93 and
sup-10 have been cloned and encode novel putative transmembrane proteins4. We cloned
sup-9 and found that it encodes a member of the TWIK-1 family of K+ channels. A rescuing
sup-9::GFP translational fusion is expressed in the body-wall muscles, the
vm1 vulval muscles, and in the male diagonal tail muscles. We have injected
sup-9 cRNA into Xenopus oocytes but have not yet detected K+ selective currents. As the gain-of-function activity of
sup-9 requires
sup-10 and
unc-93, it is possible that
sup-9 also requires these putative regulatory subunits for wild-type activity. We are currently co-expressing all three putative subunits to attempt to reconstitute a functional channel complex. We have also cloned
sup-18 and found that it encodes a novel protein. Using a series of
sup-18 coding-region truncations fused to a LacZ reporter, we have assayed the membrane topology of SUP-18. We conclude that it is most likely a single-pass transmembrane protein with an extracellular N-terminus. A
sup-18::GFP reporter is expressed in body-wall muscle and in vulval muscles, which combined with the genetic interactions of
sup-18 suggests that SUP-18 may physically interact with the SUP-9 channel. We will test this model by co-expressing SUP-9 and SUP-18 in Xenopus oocytes, as well as by using immunocytochemical approaches. 1. Greenwald, I. and Horvitz, H.R. (1980). Genetics 96, 147-164. 2. Greenwald, I. and Horvitz, H.R. (1986). Genetics 113, 63-72. 3. Levin, J. and Horvitz, H.R.(1992). Genetics 135, 53-70. 4. Levin, J. and Horvitz, H.R. (1992). Journal of Cell Biology 117, 143-155.