BK channel clustering is required for normal behavioral alcohol sensitivity in C. elegans

Abstract

The large conductance, calcium- and voltage-activated potassium channel, known as the BK channel, is one of the central proteins that mediate alcohol intoxication and tolerance across species. Although ethanol targets BK channels through direct interaction, how ethanol-mediated BK channel activation causes behavioral intoxication is poorly understood. In. C. elegans, loss of function in SLO-1, the BK channel ortholog, confers profound ethanol resistance in movement and egg-laying behaviors. Here, we show that depletion of SLO-1 channels clustered at the active zones with no change in the overall channel expression level results in locomotory resistance to the intoxicating effect of ethanol, equivalent to that of slo-1 loss-of-function mutants. Likewise, depletion of clustered SLO-1 channels in the sarcolemma and neurons leads to ethanol-resistant egg-laying behavior. By contrast, reduction in the overall SLO-1 channel level by over 70% causes only moderate ethanol resistance in movement, and minimal, if any, resistance in egg laying. Our findings strongly suggest that behavioral ethanol sensitivity is conferred by local, but not global, depression of excitability via clustered BK channels. Given that clustered BK channels are functionally coupled to, and localize near, calcium channels, ethanol may mediate its behavioral effects by targeting BK channels and their coupled calcium channels.

Figure 1

ctn-1 and erg-28 mutations confer differential resistance to the intoxicating effect of ethanol. (A) The locomotory speeds of wild-type, slo-1(eg142), erg-28(gk697770), or ctn-1(eg1167) mutant animals were measured in the presence of ethanol and their values were divided by the average speed of untreated animals. Error bars represent S.E.M. One-way ANOVA with Tukey’s post-hoc analysis (wild-type vs. erg-28: p = 0.0473, wild-type vs. ctn-1: p < 0.001, wild-type vs. slo-1: p < 0.001, slo-1 vs. ctn-1: p = 0.8136). Data points represent three independent trials of 10 animals. See Supplementary Video 1. (B) Snapshots of single animals in the presence of an intoxicating dose of ethanol show sinusoidal posture.

Figure 2

Mutation of the ctn-1 gene depletes clustered SLO-1 channels in neurons, whereas mutation of the erg-28 gene reduces the number of clustered SLO-1 channels. (A) SLO-1 localization pattern in the dorsal cord of wild-type, erg-28(gk697770), and ctn-1(eg1167) mutant animals. The slo-1(cim105[slo-1::GFP]) strain, which was generated using genome-editing, was used in the analysis. Scale bar, 5 μm. (B) The number of SLO-1 puncta representing clustered SLO-1 channels in the dorsal cord. n = 10, Sample numbers represent independent biological replicates.; F(9, 9) = 4.173, p = 0.0447, ANOVA, t(10.23) = 18, p < 0.0001, unpaired two-tailed t-test. ctn-1 mutants were excluded from the test, since we could not detect stable puncta.

Figure 3

Mutation of the ctn-1 gene depletes clustered SLO-1 channels in muscle without affecting the overall SLO-1 levels. (A,B) Representative SLO-1 localization patterns in the sarcolemma of wild-type, erg-28(gk697770), and ctn-1(eg1167) mutant animals. The right panels are zoomed-in images of the boxed areas of the left panel images. Note that ctn-1 mutants exhibit low-level fluorescent SLO-1 signals that fail to form large high-intensity clusters. Scale bar: 5 μm. Comparison of head muscle SLO-1 channel clusters between wild-type and erg-28(gk697770) animals: F(17, 23) = 1.683, p = 0.2429, ANOVA, t(9.140) = 40, p < 0.0001, unpaired two-tailed t-test. (C) Western blot analysis shows that the total SLO-1::GFP ratio is not reduced in ctn-1 mutants. SLO-1::GFP intensities in wild-type, erg-28(gk697770), and ctn-1(eg1167) were normalized to α-tubulin, and their intensities relative to wild-type are indicated. N2 represents a wild type animal that does not express GFP and serves as a negative control for Western blot with an anti-GFP antibody. The blot was probed first with anti-GFP antibody (anti-rabbit monoclonal) and then with anti-α-tubulin antibody (anti-mouse monoclonal) (Supplementary Fig. 3).

Figure 4

CTN-1-mediated SLO-1 clustering in neurons is critical for alcohol intoxication. (A,B) Muscle expression of a wild-type ctn-1 in ctn-1(eg1167) mutant animals restores SLO-1 clustering in the sarcolemma, but not the neurons in the dorsal cord. The white arrows indicate the dorsal cord. +muscle: myo-3 promoter-driven expression of ctn-1. Scale bar, 10 μm. Comparison of muscle SLO-1 channel clusters between wild-type and muscle-rescued ctn-1 mutant animals: F(29, 29) = 1.393, p = 0.3770, ANOVA, t(1.296) = 58, p = 0.2002, unpaired two-tailed t-test. (C,D) Neuronal expression of a wild-type ctn-1 in ctn-1(eg1167) mutant animals restores SLO-1 clustering at presynaptic terminals, but not sarcolemma. +neuron: rgef-1 promoter-driven expression of ctn-1. Scale bar,10 μm. Comparison of neuronal SLO-1 channel clusters between wild-type and neuron-rescued ctn-1 mutant animals: F(9, 9) = 1.406, p = 0.6197, ANOVA, t(5.405) = 18, p < 0.0001, unpaired two-tailed t-test. (E) Neuronal, but not muscular, ctn-1 expression restores normal ethanol-sensitive locomotory behavior. Locomotory speed was measured as in Fig. 1. +muscle: myo-3 promoter-driven expression of ctn-1. +neuron: H20 pan-neuronal promoter-driven expression of ctn-1. Error bars represent S.E.M. One-way ANOVA with Tukey’s post-hoc analysis (ctn-1 ethanol treated vs. ctn-1(+neuron) ethanol treated: p = 0.0145, ctn-1 ethanol treated vs. ctn-1(+muscle) ethanol treated: p = 0.8477). Data points represent three independent trials of 10 animals.

Figure 5

Mutation of the ctn-1 gene, but not the erg-28 gene, causes strong ethanol-resistant egg-laying behavior. Egg-laying behavior of wild-type, slo-1(eg142), erg-28(gk697770), or ctn-1(eg1167) mutant animals was quantified by measuring the number of eggs laid over a 60 min period in the presence or absence of ethanol. Egg laying was measured in 10 age-matched animals (30 h post L4) and repeated three times (biological replicates). The data (mean + SEM) are presented as the number of eggs laid by individual animals. One-way ANOVA with Tukey’s post-hoc analysis (wild-type N2 vs. wild-type N2 ethanol treated: ****p < 0.0001, slo-1 vs. slo-1 ethanol treated: p = 0.1174, erg-28 vs. erg-28 ethanol treated: ****p < 0.0001, ctn-1 vs. ctn-1 ethanol treated: p < 0.1517).

Figure 6

CTN-1-mediated SLO-1 clustering in neurons and egg-laying muscles contributes to ethanol-mediated suppression of egg laying behavior. (A) Muscle expression of a wild-type copy of ctn-1 in ctn-1(eg1167) mutant animals restores SLO-1 clustering at egg-laying muscles. The images represent a maximum projection of Z-stack image sections. The asterisks, arrows, and arrowheads denote the ventral nerve cord, the vulval slit, and SLO-1 clusters present in egg-laying muscle cells, respectively. The number of SLO-1 channel clusters were quantified from a region of a single muscle cell, which was not overlapped with body wall muscle or the ventral cord. Comparison of vulval muscle SLO-1 channel clusters between wild-type and muscle-rescued ctn-1 mutant animals: F(6, 6) = 1.039, p = 0.96, ANOVA, t(1.482) = 12, p < 0.1642, unpaired two-tailed t-test. WT: wild-type, +muscle: myo-3 promoter-driven expression of ctn-1. (B) Neuronal and muscular SLO-1 channel clustering contributes to ethanol-mediated suppression of egg laying. Egg laying was measured as in Fig. 5. Egg laying was measured in 10 age-matched animals (30 h post L4) and repeated three times (biological replicates). +muscle: myo-3 promoter-driven expression of ctn-1. +neuron: H20 pan-neuronal promoter-driven expression of ctn-1. Error bars represent S.E.M. One-way ANOVA with Tukey’s post-hoc analysis (ctn-1 ethanol treated vs. ctn-1(+neuron) ethanol treated: p = 0.0086, ctn-1 ethanol treated vs. ctn-1(+muscle) ethanol treated: p < 0.0001).