Mutations in the intestinal gap junction subunit innexin-16 (
inx-16) result in poor cell-to-cell communication in the intestine. This disrupts a calcium wave that flows about once every minute controlling the defecation motor program. An abnormal
inx-16 calcium wave leads to what appears to be a dietary restriction phenotype: extended lifespan and a smaller brood size laid over a longer period of time. We hypothesized that
inx-16 mutant worms exhibit this dietary restriction phenotype due to a lack of nutrient absorption. Di- and tripeptide uptake is accomplished by the transporter PEPT-1, which is dependent on the proton gradient across the apical intestinal membrane. The proton gradient itself is maintained by a sodium proton pump whose activity is regulated by calcium flux. The interrelated nature of calcium dynamics, the proton gradient, and di- and tripeptide uptake led us to predict that the
inx-16 mutation would suppress PEPT-1 mediated nutrient uptake. To delineate the genetic and phenotypic relationship between
inx-16 and
pept-1 we have created an
inx-16;
pept-1 double mutant and begun to compare the phenotypes of the single and double mutants.
pept-1 mutants' failure to absorb di- and tripeptides results in a number of salient phenotypes including a reduced brood size, a shorter body length, and increased thermotolerance that we have analyzed. Brood size comparisons reveal already more than 50% reduction in both
inx-16 and
pept-1 single mutants when compared to wild-type, whereas the
inx-16;
pept-1 double mutant strain shows a more than 80% reduced fecundity. Analyzing the developmental profiles also suggests an additive effect in the double mutant. Other parameters are different between the two single mutants. Both single mutants display thermotolerance relative to wild-type, yet
pept-1's tolerance is more profound. This magnitude difference between single mutants is mirrored in a fluorescently labeled dipeptide uptake assay.
inx-16 mutants significantly differ from wild-type worms in dipeptide uptake. However,
inx-16 mutants still absorb dipeptide in selected intestinal regions, unlike
pept-1 mutants. To learn more about the mechanistic differences between the mutants, we are conducting RT-qPCR experiments to investigate what pathways may be disrupted in these strains. Taken together, our results suggest that the dietary restriction phenotypes in
inx-16 and
pept-1 mutants are at least partially due to disruptions in different pathways.