Figure 8.

CLH-3 is not required for chloride efflux and mediates chloride influx. A, The effect of GABA_AR activation can vary from inhibitory to excitatory. When intracellular levels of chloride are low, GABA_AR activation leads to chloride influx, which inhibits via hyperpolarization or shunting inhibition; when intracellular levels of chloride are high, GABA_AR activation leads to chloride efflux, which can become excitatory. In C. elegans, the GABA_AR agonist muscimol can be used to determine if intracellular chloride levels are altered in the HSN (Tanis et al., 2009). In the wild-type, muscimol inhibits the HSN via activation of the GABA_AR UNC-49. Mutants defective in chloride extrusion have elevated intracellular chloride levels and are resistant to the inhibitory effects of muscimol. In contrast to the chloride-extruding transporters kcc-2 and abts-1, clh-3 mutants are not resistant to muscimol, indicating that they are not required for chloride efflux from the HSN. Bars represent the means ± SEM of > 5 experiments per genotype. B, C, Genetic interactions between clh-3(n995) and chloride-extruding transporters kcc-2 and abts-1. B, kcc-2 and abts-1 mutants retain fewer eggs than the wild-type. These mutations are epistatic to clh-3(n995), which leads to an accumulation of more eggs than the wild-type. Bars represent means ± SEM. n = > 36 for each genotype. For statistical analyses, single mutants are compared with wild-type and the double mutants are compared with clh-3(n995) unless otherwise indicated. C, Quantification of eggs laid at early stages (at less than the nine-cell stage) and at late stages (at more than the 400-cell stage). kcc-2 and abts-1 mutations completely suppress the egg-laying phenotypes of clh-3(n995) mutants such that the double mutants are now indistinguishable from the single mutants, with the exception of abts-1; clh-3, which lays slightly more late stage eggs than abts-1. Bars represent the proportion ± SE. n = > 120 eggs examined for each genotype. For statistical analyses, the single mutants are compared with wild-type (for all comparisons, the single mutants were indistinguishable from wild-type) and the double mutants are compared with clh-3(n995) unless otherwise indicated. For all comparisons, *p < 0.05 and ***p < 0.001. D, Model for the role of CLH-3 channels. CLH-3 normally participates in a background conductance that mediates chloride influx in the HSN. In the clh-3(lf) mutants, this conductance is absent, which makes the HSNs more excitable and leads to a hyperactive egg-laying phenotype. In the clh-3(gf) mutants, the activity of the CLH-3 channel is altered in such a way that it leads to an increase in the chloride current through the channel, which inhibits the excitability of the HSN and leads to an egg-laying-defective phenotype. Mutation of the chloride extruders kcc-2 and abts-1 increases [Cl]_i, reverses the driving force for chloride flow through the channel, reverses the effects of increasing the activity of the channel, and thereby restores egg laying. This indicates that the driving force normally promotes chloride influx through the CLH-3 channels.