Anoctamin-1 is a core component of a mechanosensory anion channel complex in C. elegans

Abstract

Mechanotransduction channels are widely expressed in both vertebrates and invertebrates, mediating various physiological processes such as touch, hearing and blood-pressure sensing. While previously known mechanotransduction channels in metazoans are primarily cation-selective, we identified Anoctamin-1 (ANOH-1), the C. elegans homolog of mammalian calcium-activated chloride channel ANO1/TMEM16A, as an essential component of a mechanosensory channel complex that contributes to the nose touch mechanosensation in C. elegans. Ectopic expression of either C. elegans or human Anoctamin-1 confers mechanosensitivity to touch-insensitive neurons, suggesting a cell-autonomous role of ANOH-1/ANO1 in mechanotransduction. Additionally, we demonstrated that the mechanosensory function of ANOH-1/ANO1 relies on CIB (calcium- and integrin- binding) proteins. Thus, our results reveal an evolutionarily conserved chloride channel involved in mechanosensory transduction in metazoans, highlighting the importance of anion channels in mechanosensory processes.

Fig. 1. Mechanical stimulation evokes ANO1-dependent calcium increases in CEM neurons.

a Micrograph showing four CEM neurons in the head of an adult male worm. Scale bars, 10 µm. b Nose touch evoked a calcium increase in CEM. Left: representative time-lapse images of GCaMP5.0- based calcium responses in CEM induced by nose touch; Right: calcium response indicated by fluorescence changes of GCaMP5.0. The arrows indicate the application of mechanical force (15 μm displacement). c The touch-evoked calcium responses in CEM were dependent on chloride ions and were abolished by NFA. Left: calcium responses; Right: maximum ΔF/F₀ changes. Solid lines show the average fluorescence changes and the shading indicates SEM. P values were calculated using the Brown-Forsythe and Welch ANOVA tests. d Chloride transporter mutants affected the nose touch-evoked calcium increases in CEM. Left: maximum ΔF/F₀ changes; Right: calcium responses. P values were calculated using the Brown-Forsythe and Welch ANOVA tests. e, f The absence of ANOH-1 or BEST-2 significantly reduced the mechanically activated chloride channels in CEM. Calcium responses (f); Maximum ΔF/F₀ changes (e). P values were calculated using the Kruskal-Wallis test. g Transgenic expression of either nematode or human anoctamin-1 genes rescued the nose touch-evoked calcium increases in CEM of anoh-1(tm4762) mutant male worms. Left: calcium responses; Right: maximum ΔF/F₀ changes. The mutations of anoh-1(K588A) and ANO1(K645A) will be further elucidated in the subsequent sections. P values were calculated using the Kruskal-Wallis test. Day 2 adult male animals were used in these experiments. The numbers of independent assays are indicated in each column of the panel. Each dot represents 1 animal. Data are presented as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 2. ANOH-1 is required for touch-evoked calcium responses in ASJ neurons.

a Expression pattern of anoh-1 in hermaphroditic worm. Scale bars, 10 µm. Repeated independently with 10 worms. b The nose touch-induced calcium increases in ASH. Left: calcium responses; Right: maximum ΔF/F₀ changes. Mechanical stimulation: 20 µm displacement. c MRCs in ASH require DEG-1 but not ANOH-1. Left: sample traces; Right: peak MRC amplitudes. Mechanical stimulation: 15 µm displacement. P values were calculated using the Brown-Forsythe and Welch ANOVA tests. d Micrograph showing two ASJ neurons of an adult hermaphroditic worm. Scale bars, 10 µm. Repeated independently with 10 worms. e Nose touch evoked a calcium increase in ASJ. Left: representative time-lapse images of GCaMP6s-based calcium responses in ASJ induced by nose touch; Right: calcium response. Mechanical force: 20 μm displacement. f Mechanical stimulation-induced calcium increases in ASJ. Left: calcium responses; Right: maximum ΔF/F₀ changes. The red arrow indicates the application of mechanical force (20 μm displacement). The mutations of anoh-1(K588A) and ANO1(K645A) will be further elucidated in the subsequent sections. P values were calculated using the Kruskal-Wallis test. g Histamine-induced calcium responses in ASJ with the transgenetic expression of HisCl1 channel. Left: calcium responses; Right: maximum ΔF/F₀ changes. P values were calculated using the Mann-Whitney test. h Histamine-induced calcium responses in CEM with the transgenetic expression of HisCl1 channel. Left: calcium responses; Right: maximum ΔF/F₀ changes. P values were calculated using the Welch’s t test. i Histamine-induced calcium responses in CEM in chloride transporter mutants. Left: calcium responses; Right: maximum ΔF/F₀ changes. P values were calculated using the Brown-Forsythe and Welch ANOVA tests. The numbers of independent assays are indicated in each column of the panel. Each dot represents 1 animal. Data are presented as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 3. ANOH-1 is required for nose touch behavior.

a CEM neurons were essential for the nose-touch behaviors of male worms. Each dot represents 1 independent assays, and 10 animals were used for each assay. P values were calculated using Brown-Forsythe and Welch ANOVA test. b ANOH-1 was essential for avoidance behaviors in response to nose touch. Each dot represents 1 independent assays, and 10–15 animals were used for each assay. P values were calculated using Kruskal-Wallis test. The mutations of anoh-1(K588A) and ANO1(K645A) will be further elucidated in the subsequent sections. Data are presented as mean ± SEM. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 4. ANOH-1 mediates Ca²⁺-independent mechanoreceptor currents in ASJ neurons.

a Schematic illustration of mechanoreceptor currents (MRCs) recording in ASJ neurons from a dissected worm. A glass probe with a diameter of about 10 μm was placed near the cilium of the ASJ neuron and mechanical force was applied by a piezo actuator. b Representative MRC in ASJ evoked by mechanical stimulation with 15 μm displacement. Holding potential: −70 mV. c Latency of MRCs in ASJ. Left: sample trace. Right: latency values. Mechanical stimulation: 15 μm displacement. Holding potential: −70 mV. d MRCs in ASJ. Left: sample trace. Right: the amplitude of MRCs. Mechanical stimulation: 15 μm displacement. Holding potential: −70 mV. P values were calculated using Kruskal-Wallis test. e MRCs in ASJ were independent of extra- and intracellular Ca²⁺, and blocked by NFA and T16A_inh-A01. Left: sample traces. Right: peak MRC amplitudes. Holding potential: −70 mV. P values were calculated using Kruskal-Wallis test. f MRCs in ASJ neurons were dependent on both intracellular and extracellular chloride concentrations. Left: representative traces. The cell membrane was initially voltage-clamped at −50 mV, 0 mV, and 50 mV, respectively, and the displayed voltages have been corrected posthoc for liquid junction potentials (LJPs). Middle: I–V relationship of MRCs in ASJ. Peak current values were used here and throughout the manuscript. Right: the reversal potentials of MRCs. Mechanical stimulation: 15 μm displacement. P values were calculated using Brown-Forsythe and Welch ANOVA tests. Day 2 adult hermaphroditic animals were used in these experiments. Each dot represents 1 animal. Data are presented as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 5. MRCs recorded in the isolated ASJ neurons.

a Schematic illustration of MRCs recording in the isolated ASJ neurons. b An isolated ASJ neuron was labeled by Ptrx-1::mCherry. Scale bar, 10 μm. Repeated independently with 10 worms. c A representative MRC recorded from an isolated ASJ neuron. Mechanical stimulation: 4 μm displacement. Holding potential: −70 mV. d MRCs recorded in isolated ASJ neurons were dependent on both intracellular and extracellular chloride concentrations. Left: representative traces of MRCs. The cell membrane was initially voltage-clamped at −50 mV, 0 mV, and 50 mV, with the displayed voltages corrected posthoc for liquid junction potentials (LJPs). Middle: I–V relationship of MRCs. Right: the reversal potentials of MRCs. Mechanical stimulation: 4 μm displacement. Each dot represents 1 single cell. P values were calculated using Brown-Forsythe and Welch ANOVA tests. Data are presented as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 6. Ectopic expression of nematode ANOH-1 or human ANO1 confers mechanosensitivity to ASK neurons.

a MRCs of wild-type ASK neurons or ASK neurons ectopically expressing nematode ANOH-1 or human ANO1. Left: sample traces; Right: peak MRC amplitudes. Mechanical stimulation: 15 μm displacement. Holding potential: −70 mV. P values were calculated using Kruskal-Wallis test. b MRCs in ASK ectopically expressing ANOH-1. Left: sample traces; Right: peak MRC amplitudes. P values were calculated using Kruskal-Wallis test. c MRCs recording in ASK neurons ectopically expressing ANOH-1. Left: representative traces of MRCs. The cell membrane was initially voltage-clamped at −50 mV, 0 mV, and 50 mV, and the displayed voltages were subsequently corrected for liquid junction potentials (LJPs). Middle: I-V relationship of MRCs. Right: the reversal potential of MRCs. Mechanical stimulation: 15 μm displacement. P values were calculated using Brown-Forsythe and Welch ANOVA tests. Mutations in the predicted pore region of ANOH-1 abrogated mechanosensitivity of ASK neurons with ectopic expressing of ANOH-1. d Sequence alignment of the putative pore region of ANOH-1 and its homologs. The positively charged residues K and R between TM5-TM6 regions are highly conserved among ANOH-1 and its homologs. Homology alignment was performed using the MEGA11. e No MRC was recorded in ASK neurons ectopically expressing ANOH-1 with either K588E or K588A mutations. Left: sample traces. Right: peak MRC amplitudes. Mechanical stimulation: 15 µm displacement. Holding potential: −70 mV. f Membrane topology of ANOH-1 and the location of positively charged K588 residue. P values were calculated using Kruskal-Wallis test. Day 2 adult hermaphroditic animals were used in these experiments. Each dot represents 1 animal. Data are presented as mean ± SEM. ns not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 7. CIB proteins are required for the mechanosensory function of ANOH-1/ANO1.

a The touch-evoked calcium increases in CEM. Each dot represents 1 animal. P values were calculated using Kruskal-Wallis test. b Nose touch induced reversals of Day 2 adult male animals. Each dot represents 1 independent assays, and 10 ~ 15 animals were used for each assay. P values were calculated using Brown-Forsythe and Welch ANOVA tests. c The touch-evoked calcium increases in ASJ. Expression of either nematode CALM-1 or human CIB2, but not human CIB3, in ASJ neurons was able to rescue the defects observed in the calm-1 mutants. Left: calcium responses; Right: maximum ΔF/F₀ changes. Mechanical stimulation: 15 μm displacement. Each dot represents 1 animal. P values were calculated using Kruskal-Wallis test. d MRCs in ASJ. Left: calcium responses; Right: maximum ΔF/F₀ changes. Each dot represents 1 animal. P values were calculated using Kruskal-Wallis test. e MRCs were detected in the ASK neurons of calm-1 mutant worms that ectopically expressed nematode ANOH-1 with CALM-1 or human ANO1 with CIB2. Left: sample traces; Right: peak MRC amplitudes. Each dot represents 1 animal. P values were calculated using Kruskal-Wallis test. f Co-IP experiments with HEK293T cells showing the direct binding of exogenously expressed nematode ANOH-1 with CALM-1, or human ANO1 with CIB2. Repeated for twice. g ANOH-1/ANO1 acts as a core component of a mechanosensory anion channel complex. The CIB and ankyrin proteins are likely auxiliary components of the mechanotransduction channel complex. Ectopic expression of ANOH-1 or human ANO1 channels confers mechanosensitivity to mechanically insensitive neurons through a Calm-1/CIB2-dependent mechanism, suggesting that the role of Anoctamin 1 in mechanotransduction is evolutionarily conserved. Data are presented as mean ± SEM. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.