unc-79 and unc-80 encode large proteins necessary for proper localization of the NCA ion channel (Humphrey et al., 2007; Jospin et al., 2007; Yeh et al., 2008). unc-79 and unc-80 also show genetic interactions with mutants in the ENaC ion channel unc-8 (Rajaram et al., 1999). Loss-of-function mutants in unc-8 were reported to suppress the fainter and anesthetic sensitivity phenotypes of unc-79 and unc-80. Additionally, the exceptional allele unc-8(n491 n1193) was reported to have a fainter phenotype on its own. Thus, it seemed possible that unc-79 and unc-80 regulate both the UNC-8 and NCA channels. To investigate this further, we reexamined interactions of unc-8 and unc-80 in more detail. We conclude that unc-8 does not suppress the fainter phenotype of unc-80.

The fainter phenotype of unc-8(n491 n1193) is in fact due to a background mutation in unc-80. First, we genetically separated the fainter phenotype of MT2612 unc-8(n491 n1193) from the unc-8 chromosome, using bli-6 dpy-20 to balance unc-8. We isolated a fainter mutant that did not carry unc-8(n491 n1193) and an unc-8(n491 n1193) mutant that did not carry the fainter phenotype. The fainter phenotype was unlinked to unc-8(n491 n1193). Second, we found that the fainter mutation in this strain failed to complement mutations in unc-80. Third, we sequenced unc-80 in this mutant (named ox374) and found a C to T change that leads to a premature stop at Q2811. The unc-80(ox374) single mutant is a fainter indistinguishable from other unc-80 mutants. The unc-8(n491 n1193) single mutant appears to be grossly wild-type, similar to other unc-8(lf) mutants. The unc-8(n491 n1193); unc-80(ox374) double mutant is a fainter, like the unc-80(ox374) single mutant. The genotypes of the unc-8 and unc-80 loci were confirmed in all single and double mutants by sequencing.

We were surprised that the unc-8(n491 n1193); unc-80(ox374) double mutant is a fainter since the unc-8(e15 lb145) deletion mutant was reported to suppress the fainter and anesthetic phenotypes of unc-79 and unc-80. One possibility to explain this discrepancy is that unc-8(n491 n1193) carries an E552K missense mutation and may not be as strong as a null. To test this, we built a double mutant between unc-8(e15 lb145) and unc-80(ox330), an early stop mutant. Similar to the unc-8(n491 n1193); unc-80(ox374) double, we observed no suppression of the fainter phenotype in an unc-8(e15 lb145); unc-80(ox330) double mutant. We are in the process of reexamining the anesthetic phenotypes of these double mutants.