Worm Breeder's Gazette 13(3): 77 (June 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Maintenance of osmotic balance is a vital cell function. Cell volume and membrane potential both depend on proper ion distribution across the plasma membrane. Specialized functions of some cell types, such as neuronal signalling and trans-epithelial transport are also critically dependent on electrochemical gradients. Mutational analysis can be used to identify new ion channel genes and to examine the relationship between ion channel structure and function. We devised a drug-resistance screen which identifies such genes in C. elegans. Both unc-8 and its newly-identified dominant suppressor locus sup-40 (LGI) can mutate to confer increased survival after exposure to a toxic level of nordihydroguairetic acid (NDG), a non-specific lipoxygenase inhibitor shown to block opening of Aplysia S-K+ channels. (1) A separate class of NDG-resistant mutants, identifying at least two other genes, was found, which are behaviorally normal but which exhibit excess liquid in the pseudocoelom of adults, abnormally swollen coelomocytes, and leaky sterility.
Dominant mutations of the unc-8 gene cause defective locomotion. (2) We observed that unc-8 ( e15 )and n491 mutations also cause motorneuron swelling, suggesting an osmotic defect. Dominant missense mutations in three previously isolated genes, deg-1 , mec-4 and mec-10 (5), also cause neuronal swelling. deg-1 (3), mec-4 (4), and mec-10 (5) are members of a gene family with significant sequence (6,7) and functional (8) homologies to the amiloride-sensitive epithelial sodium channel of mammals. We also found that both the uncoordinated locomotion and swollen motorneuron phenotypes of unc-8 mutants are fully suppressed by mutations of the mec-6 gene. mec-6 mutations also suppress the dominant cell-swelling phenotype caused by deg-1 , mec-4 ,and mec-10 mutations . (3, 5) We found as well that, like the deg-1 locus, unc-8 is a dispensable gene (9) and exhibits interallelic interactions suggesting that its product functions as a multimer (10). Furthermore, we isolated a dominant suppressor mutation of unc-8 ( n491 )which cross-suppresses deg-1 ( u38 ).Thus, by genetic criteria, unc-8 appears to encode a new member of the deg-1 family of ion channel components.
We also identified a different unc-8 suppressor locus, sup-40 (LGI), that can mutate to dominantly suppress the unc-8 motorneuron defect. Thus, sup-40 appears to regulate motorneuron ion channel function. sup-40 ( lb130 )also confers three recessive phenotypes: NDG resistance, swelling of hypodermal nuclei and almost complete disruption of oogenesis. Those oocytes which do form are abnormally large, misshapen, and frequently burst apart, as if under internal pressure. These recessive phenotypes suggest that sup-40 is involved in control of membrane permeability in hypodermis and oocytes as well as in motorneurons. mec-6 mutations do not suppress the recessive sup-40 phenotypes, nor does the sup-40 ( lb130 )cross-suppress deg-1 ( u38 ).
The drug-resistance of unc-8 dominant mutations and of the sup-40 ( lb130 )mutation suggest that NDG may deleteriously alter the functioning of the wild-type channel. The biochemical mechanism of NDG toxicity in C elegans is not yet known, however, NDG can act as a lipoxygenase inhibitor in other systems. It is thus possible that lipoxygenase products of arachidonic acid, such as HPETE or its metabolites may affect channel function, as they have been reported to do at S-type K+ channels of Aplysia.(1) NDG does affect the C. elegans nervous system: brief exposure of wild-type worms to NDG induces egg-laying, and unc-8 dominant mutants display significantly elevated basal egg-laying as compared to wild-type. This phenotype is also sup-40 -suppressible.Thus, the unc-8 ion channel and its sup-40 partner are components of egg-laying as well as locomotory circuitry. Although many questions remain about the actions of NDG in C. elegans, we have found NDG-resistance to be a useful criterion for expanding the collection of mutations affecting neuronal signalling and cell membrane
permeability.
2. Genetics 77:71.
3. Nature 345:410.
4. Nature 349:588.
5. Nature 367:467.
6. Nature 367:463.
7. Nature 361:504
8. Nature 367:467.
9. Genetics 113:821.
10. WBG 11(4):104.