Rare altered-function mutations in the genes
unc-93,
sup-9, and
sup-10 result in the abnormal regulation of muscle contraction. These mutants move sluggishly and are unable to lay eggs. Loss-of-function mutations in any of these three genes completely suppress the phenotypes caused by any of the altered-function mutations, suggesting that all three genes act at the same step, possibly by encoding subunits of a protein complex. We have shown that
sup-9 encodes a two-pore K+ channel subunit with similarity to the mammalian Two-pore Acid Sensitive K+ channels TASK-1 and TASK-3.
unc-93 and
sup-10 encode novel putative transmembrane proteins that likely serve as regulatory subunits of SUP-9.
sup-18 may encode a positive regulator of the
sup-9/sup-10/unc-93 channel complex.
sup-18(lf) mutations fully suppress the Unc and Egl defects of
sup-10(gf) mutants, while only partially suppressing those of
unc-93(gf) or
sup-9(gf) mutants, suggesting that
sup-18 may be preferentially required for the
sup-10(gf) activity. In addition, we have found that
sup-18(lf) mutations also only partially suppress the weaker defects of partial
sup-10(lf) double mutants with
unc-93(gf) mutations, indicating that the partial suppression of
unc-93(gf) defects by
sup-18(lf) is not caused by the greater severity of
unc-93(gf) defects. We previously cloned
sup-18 and found that it encodes a type-one transmembrane protein, the cytoplasmic domain of which contains a nitroreductase domain. We found that the strong loss-of-function allele
sup-18(
n1010) mutates a highly conserved serine to an asparagine within the nitroreductase domain. The equivalent serine in a nitroreductase from Thermus thermophilus (Ttnox) contacts a tightly-bound FMN cofactor. We found that recombinant TtNOX carrying a serine-to-asparagine mutation at this site had severely reduced NADH oxidase activity in vitro, consistent with the hypothesis that SUP-18 has an enzymatic activity in vivo. To explore the mechanisms by which
sup-10(gf) and
unc-93(gf) mutations activate
sup-9, we have analyzed an unusual
sup-9 mutant.
sup-9(
n1435) suppresses fully the Unc and Egl defects of
sup-10(gf) mutants but only weakly those of
unc-93(gf) mutants, unlike null mutations in
sup-9 which fully suppress defects in both mutants. We hypothesized that
sup-9(
n1435) may be insensitive to
sup-18 and therefore displays the same differential suppression as
sup-18(lf) mutations. Consistent with this model, the partial suppressive effects of
sup-9(
n1435) and
sup-18(lf) towards
unc-93(gf) defects were not additive in the triple mutant. The
sup-9(
n1435) mutation leads to a serine-to-phenylalanine substitution in the presumptive C-terminal cytoplasmic domain of
sup-9. Using site-directed mutagenesis, we identified another residue in this domain that is required for
sup-18- but not for
unc-93-dependent activation. In addition, we have found that overexpression of
sup-18 in a
sup-10(gf) but not in an
unc-93(gf) mutant enhances the severity of the Unc defects. Together, these results support a model in which the
sup-10(gf) mutation acts with the
sup-18 nitroreductase to activate the
sup-9 channel through a mechanism that is distinct from that of
unc-93(gf) mutations.