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. 2014 Jan 15;34(3):764-75.
doi: 10.1523/JNEUROSCI.3112-13.2014.

The Voltage-Gated Anion Channels Encoded by clh-3 Regulate Egg Laying in C. Elegans by Modulating Motor Neuron Excitability

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The Voltage-Gated Anion Channels Encoded by clh-3 Regulate Egg Laying in C. Elegans by Modulating Motor Neuron Excitability

Robyn Branicky et al. J Neurosci. .
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Abstract

CLC-2 is a hyperpolarization-activated, inwardly rectifying chloride channel. Although the properties of the CLC-2 channel have been well characterized, its function in vivo is not well understood. We have found that channels encoded by the Caenorhabditis elegans CLC-2 homolog clh-3 regulate the activity of the spontaneously active hermaphrodite-specific neurons (HSNs), which control the egg-laying behavior. We identified a gain-of-function mutation in clh-3 that increases channel activity. This mutation inhibits egg laying and inhibits HSN activity by decreasing its excitability. Conversely, loss-of-function mutations in clh-3 lead to misregulated egg laying and an increase in HSN excitability, indicating that these channels modulate egg laying by limiting HSN excitability. clh-3-encoded channels are not required for GABAA-receptor-mediated inhibition of the HSN. However, they require low intracellular chloride for HSN inhibition, indicating that they inhibit excitability directly by mediating chloride influx. This mechanism of CLH-3-dependent modulation may be conserved in other neurons in which the driving force favors chloride influx.

Figures

Figure 1.
Figure 1.
The n995 mutation is a gain-of-function mutation in clh-3. A, Gene structure of the clh-3 locus. clh-3 encodes two isoforms; the a isoform is shown on top and the b isoform on bottom. The n995/n996 mutation is a T to C substitution resulting in a V566A substitution in CLH-3a and a V495A substitution in CLH-3b. B, Alignment of membrane embedded α-helices O–R of CLH-3a and CLH-3b with human (h), Cyanidioschyzon merolae (Cm), Arabidopsis thaliana (At), and Escherichia coli (Ec) CLC proteins. The n995 mutation substitutes an alanine for a highly conserved valine residue (highlighted) in the P-helix. C, DIC images of young adult worms all at the same age. Arrows indicate embryos; arrowheads indicate late-stage embryos (>400 cells). clh-3(n995) mutants and wild-type animals overexpressing clh-3(n995) engage in egg laying less frequently than the wild-type, which leads to the accumulation of twice as many eggs as the wild-type and clh-3(ok763) mutants, many of which reach >400 cells before they are laid. DF, Quantification of number of eggs retained in the uterus of adults. Bars represent the means ± SEM. ***p < 0.001 compared with N2 or as indicated; n>36 for each genotype or as indicated. D, clh-3(n995) is a dominant mutation that leads to the accumulation of eggs. E, Overexpression of both wild-type and mutant clh-3 leads to the accumulation of eggs. F, Expression of the clh-3b isoform, but not the clh-3a isoform, in the HSN leads to the accumulation of eggs. Introducing a stop codon early in the clh-3b coding sequence abrogates the effect, indicating that it is dependent on production of the CLH-3b protein. More than 10 independent transgenic lines were examined for each construct and one representative line is shown. n = > 24 for each transgenic line.
Figure 2.
Figure 2.
CLH-3 regulates egg laying by regulating HSN function. A, Pharmacology assay for HSN function. The HSNs release serotonin (5HT), which directly stimulates the vulval muscles (VM) and promotes egg laying. In the wild-type, egg laying can be promoted by exogenous 5HT as well as imipramine (imi) and fluoxetine (fluo), which inhibit the reuptake of 5HT. In mutants lacking the HSN or HSN function, egg laying can only be promoted by exogenous 5HT (Trent et al., 1983). In clh-3(n995gf) mutants and wild-type worms overexpressing clh-3(n995gf), egg laying can be robustly stimulated by exogenous 5HT, but significantly less so by imipramine or fluoxetine, indicating a defect in HSN function. clh-3(ok763 and ok768) mutants lay more eggs in the presence of imipramine or fluoxetine than 5HT. Bars represent the means ± SEM. n = > 36 for each genotype and treatment. For statistical analyses, the effects of fluo and imi treatment are compared with the effect of 5HT treatment on that genotype. B, Comparison of egg-laying rates in the presence and absence of food. In the wild-type, egg laying is inhibited in the absence of food. Although clh-3(n995gf) mutants lay fewer eggs than the wild-type in the presence of food, clh-3(ok763 and ok768) mutants lay more eggs than the wild-type in the absence of food. Bars represent the means ± SEM. n = > 15 for each genotype on food and n = > 36 off food. For statistical analyses, mutants are compared with the wild-type under the same condition. C, Comparison of egg laying in M9 buffer. In the wild-type, egg laying is inhibited by high osmolarity. Egg laying is similarly inhibited in clh-3 mutants. Bars represent the means ± SEM. n = > 22 for each genotype. For statistical analyses, mutants are compared with the wild-type. D, 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). clh-3(n995gf) mutants lay fewer eggs than the wild-type at early stages and more eggs than the wild-type at late stages; clh-3(ok763 and ok768) mutants lay more eggs than the wild-type at early stages. Bars represent the proportion ± SE of the proportion. n = > 100 eggs examined for each genotype. For all comparisons, *p < 0.05, **p < 0.001, and ***p < 0.001.
Figure 3.
Figure 3.
Functional properties of wild-type and V566A CLH-3a channels. A, Representative current traces. B, Current-to-voltage relationships. *p < 0.01 compared with wild-type CLH-3a. Statistical analyses were performed only at voltages more hyperpolarized than the activation voltage of wild-type channels. C, Activation voltages. *p < 0.0001 compared with wild-type CLH-3a. D, Examples of whole-cell current traces of wild-type and V566A CLH-3a channels. Membrane potential was held at 0 mV and then stepped to +60 mV for 1–6 s before currents were activated by a 1 s hyperpolarizing step in membrane voltage to −100 mV. Cells were at 0 mV for 10 s after each hyperpolarizing voltage step. E, Time dependency of pre-depolarization at +60 mV on peak wild-type and V566A current amplitude at −100 mV. Peak current amplitude was measured between 100 and 200 ms and between 850 and 950 ms after onset of the −100 mV test pulse in wild-type CLH-3a and the V566A mutant, respectively. *p < 0.01 compared with wild-type CLH-3a. Values shown in B, C, and E are means ± SEM. n = 5–8.
Figure 4.
Figure 4.
Functional properties of wild-type and V495A CLH-3b coexpressed with either functional or kinase dead (KD) GCK-3. A, Representative current traces. B, C, Current-to-voltage relationships. *p < 0.001 and **p < 0.01 compared with channels coexpressed with KD GCK-3. Statistical analyses were performed only at voltages more hyperpolarized than the activation voltage of channels coexpressed with GCK-3. D, Activation voltages. *p < 0.001 compared with wild-type CLH-3b coexpressed with KD GCK-3; **p < 0.0001 compared with channels expressed with KD GCK-3. E, The 50% rise times, defined as the time required for whole-cell current to reach 50% activation when membrane voltage is stepped from 0 mV to −100 mV for 1 s. *p < 0.001 compared with channels coexpressed with KD GCK-3; **p < 0.00001 compared with wild-type channels coexpressed with GCK-3. Values shown in BE are means ± SE. n = 4–6.
Figure 5.
Figure 5.
Calcium imaging reveals that CLH-3 inhibits HSN activity. A, Sample traces. The HSNs exhibit spontaneous rhythmic activity (Zhang et al., 2008). Most clh-3(n995gf) mutants have greatly reduced HSN activity, whereas some retain wild-type levels. B, C, Quantification of calcium spike parameters. The average number of spikes/minute and average percentage ratio change of each spike was determined for each recording. Each point represents the value obtained from one recording obtained from one animal. n = > 13 recordings, which corresponds to 145 spikes for clh-3(n995) and > 750 spikes for the other genotypes. Mutants were compared with the wild-type assayed at the same time. The clh-3(n995gf) mutation significantly reduces the spike frequency. None of the mutations alter spike parameters such as ratio change (as shown), area of spike, rise time, or decay time (data not shown).
Figure 6.
Figure 6.
CLH-3 regulates the temporal pattern of egg laying. A, Schematic representation of the temporal pattern of egg laying. Animals alternate between active periods during which eggs are laid and inactive periods during which no eggs are laid (Waggoner et al., 1998). BF, Histograms of intervals between egg-laying events. Intervals are plotted on a natural log scale on the x-axis and relative frequencies are plotted on the y-axis. The graphs show a bimodal distribution, with the first peak corresponding to the intervals between events in the active periods (the intracluster intervals) and the second peak corresponding to the intervals between active periods (the intercluster intervals). Intervals <400 s were considered intracluster intervals and intervals > 400 s were considered intercluster intervals. Vertical lines indicate median intervals for each peak; dashed lines indicate the median values for the wild-type; solid lines indicate the median values for the mutant in each panel. The egl-1 mutation both shortens the intracluster intervals (p = 0.00048) and lengthens the intercluster intervals (p < 0.000001). clh-3 mutations mainly affect the intracluster intervals: in clh-3(n995gf) mutants, the intervals between egg-laying events in the cluster are increased (p = 0.00142), whereas in clh-3(ok768 and ok763) mutants, the intervals are decreased (p = 0.01663 and p = 0.00062). For the long intervals, the p-values for DF are p = 0.00093, p = 0.0702, and p = 0.29785, respectively. Sample sizes and other quantifications are shown in Table 1.
Figure 7.
Figure 7.
Channel rhodopsin experiments reveal that CLH-3 inhibits HSN excitability. A, The egg-laying response in the wild-type is dependent on both the intensity of the light stimulus and the duration of the light stimulus. Bars represent means ± SEM. B, The response to 5 s, 1 mW/mm2 blue light stimulation is dependent on the presence all-trans retinal, the cofactor for CHR2. Bars represent means ± SEM. More than nine stimulations were tested for each condition and genotype. C, Histogram of eggs laid per 5 s, 1 mW/mm2 blue light stimulation. Number of eggs laid per stimulation is plotted on the x-axis and the relative frequency is plotted on the y-axis. In clh-3(n995gf) mutants, stimulations more frequently do not lead to egg-laying events, whereas in clh-3(ok768 and ok763) mutants, stimulations more frequently do lead to egg-laying events, including the laying of more than one egg. n = > 42 stimulations/genotype. D, Quantification of number of eggs laid/stimulation in the presence of all-trans retinal. Bars represent means ± SEM. n = 18 stimulations for egl-1 and > 42 stimulations for the other genotypes. The response to blue light stimulation requires the HSN because egl-1 mutants do not respond. clh-3(n995gf) mutants lay fewer eggs per stimulation, whereas clh-3(ok768 and ok763) mutants lay more eggs. **p < 0.01; ***p < 0.001.
Figure 8.
Figure 8.
CLH-3 is not required for chloride efflux and mediates chloride influx. A, The effect of GABAAR activation can vary from inhibitory to excitatory. When intracellular levels of chloride are low, GABAAR activation leads to chloride influx, which inhibits via hyperpolarization or shunting inhibition; when intracellular levels of chloride are high, GABAAR activation leads to chloride efflux, which can become excitatory. In C. elegans, the GABAAR 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 GABAAR 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.

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