Genetic data suggest that
unc-8 IV encodes a subunit of a Na+ channel homologous to the passive leak Na+ channels subserving transepithelial salt and water transport in mammals. Physiological, biochemical, and genetic experiments indicate that the ion channel is a multi-subunit structure, of which two mammalian components have been molecularly identified: ENaC's and CFTR (Stutts et. al., Science 269:847). To identify other genes encoding proteins with important roles in ion channel function, extragenic
unc-8 suppressor and enhancer mutations were sought. Two
unc-8 suppressor loci,
sup-40 I and
sup-41 IV were described previously (Shreffler et. al. Genetics 139:1261). We describe here gene interactions of two new
unc-8 supressor loci,
sup-42(
lb88) X and
sup-43(
lb141) II, and an enhancer locus,
enu-2(
lb140). Sup-43
(lb141) is an unusual allele specific suppressor of
unc-8(
e49). Single mutants of either
sup-43(
lb141) or
unc-8(
e49) express a coiler Unc phenotype, while the double mutant
sup-43(
lb141);
unc-8(
e49) behaves as wild-type. These three mutations were initially isolated by the sole criterion of amelioration or exacerbation of
unc-8 locomotion defects, however, each displays unselected cell swelling phenotypes which further suggest an effect on membrane permeability:
sup-42(
lb88) causes swelling of embryonic cells, while
sup-43(
lb141) and
enu-2(
lb140) cause vacuoles within body wall muscle, similar in appearance to those caused by
unc-105(
n490). Waterston et. al. have shown that
unc-105 is a member of the ENaC gene family. The phenotypic similarities between
sup-43(
lb141),
enu-2(
lb140) and
unc-105(
n490) are underscored by the observation that each exhibits striking synthetic lethality in combination with a fourth mutation,
ndg-4(
lb108). Ndg-4
(lb108) confers strong resistance to nordihydroguairetic acid (NDG), a non-specific lipoxygenase inhibitor; dominant
unc-8 mutations are moderately NDG resistant. Strikingly, many
unc-105(
n490);
ndg-4(
lb108) mutant embryos are severely vacuolated, while embryonic vacuoles are not observed in either single mutant. While the biochemical basis of
ndg-4(
lb108) drug resistance is not yet understood, interactions between
ndg-4(
lb108) and mutations in three different genes causing vacuoles within body wall muscle strongly suggest some commonality of action. Interactions with
ndg-4 are particularly intriguing in the light of reports of effects of lipo- and epoxygenase inhibitors on CFTR (e.g., Kersting et. al. PNAS 90:4047). We speculate that
unc-8 suppressor mutations may act by up-regulating an endogenous eicosanoid inhibitor of ENaC activity. We are currently analysing a novel
unc-8 suppressor strain which expresses a pale egg phenotype identical to that of
ndg-4. The gene expression patterns inferred from single and double mutant phenotypes in this and previous work suggest that overlapping sets of gene products are available for construction of Na+ leak channels in motorneurons, and body wall muscle, and early development. Phenotypic effects are exerted in motorneurons by
unc-8(dom.),
sup-40-43, and
mec-6(l-o-f), in body wall muscle by
unc-8(dom. or null),
sup-43(
lb141),
enu-2(
lb140),
unc-105(
n490), and
ndg-4(
lb108), and in developing eggs or embryos by
sup-40(
lb130),
sup-42(
lb88),
sup-43(
lb141),
enu-2(
lb140),
unc-105(
n490), and
ndg-4(
lb108).