Functional analysis in a model sea anemone reveals phylogenetic complexity and a role in cnidocyte discharge of DEG/ENaC ion channels

Abstract

Ion channels of the DEG/ENaC family share a similar structure but serve strikingly diverse biological functions, such as Na⁺ reabsorption, mechanosensing, proton-sensing, chemosensing and cell-cell communication via neuropeptides. This functional diversity raises the question of the ancient function of DEG/ENaCs. Using an extensive phylogenetic analysis across many different animal groups, we found a surprising diversity of DEG/ENaCs already in Cnidaria (corals, sea anemones, hydroids and jellyfish). Using a combination of gene expression analysis, electrophysiological and functional studies combined with pharmacological inhibition as well as genetic knockout in the model cnidarian Nematostella vectensis, we reveal an unanticipated role for a proton-sensitive DEG/ENaC in discharge of N. vectensis cnidocytes, the stinging cells typifying all cnidarians. Our study supports the view that DEG/ENaCs are versatile channels that have been co-opted for diverse functions since their early occurrence in animals and that respond to simple and ancient stimuli, such as omnipresent protons.

Fig. 1. Molecular phylogenetic tree of the DEG/ENaC channel superfamily with A. queenslandica as an outgroup.

a The tree was constructed with the IQ-Tree software (see Materials and Methods). The branches of some clades are collapsed. Asterisks above and below branches denote a support value > 65% for the three different methods calculated in IQTREE and bayesian posterior probabilities >0.65 calculated in phylobayes, respectively. b Schematic representation of the phylogenetic relationship of the main groups of species selected for the sampling of their DEG/ENaCs.

Fig. 2. In situ Hybridization of selected NeNaCs at three different developmental stages in N. vectensis.

The expression is noticeable by the blue stain (NBT/BCIP crystals). Oral end of the animals is to the right for each panel. dpf days post-fertilization. Scale bars: 100 µm.

Fig. 3. NeNaC2 is a proton-gated ion channel.

a Representative trace of NeNaC2 currents elicited by increasingly more acidic pH (yellow bars). Note the biphasic current that appeared below pH 5.5. Right. b Concentration–response curve (n = 9). c Representative trace from NeNaC2-expressing oocytes injected with EGTA. d I/V plot revealing E_rev = +46 mV for Na⁺ (black circles) and E_rev = −30 mV for Ca²⁺ (black triangles). e I/V plot revealing E_rev = +46 mV for Na⁺ (black circles) and E_rev = +6 mV for K⁺ (black circles). Error bars represent S.D. f Representative current traces of NeNaC2 in the presence of increasing concentrations of amiloride or diminazene (green and turquoise bars, concentrations in µM); NeNaC2 was activated with pH 6.0. g Concentration–response curves. Error bars represent S.D. h Representative current traces of NeNaC2 WT, NeNaC2H141A, and of water-injected control oocytes. i Alignment of the region close to β1 of rASIC1a, hASIC2, hASIC3, NeNaC2, and NeNaC23. j Mean current amplitudes of NeNaC2 (yellow dots, n = 8) and NeNaC2H141A (black squares, n = 6). Current amplitudes were reduced for the mutant, but apparent proton affinity was similar. Error bars represent S.E. Concentration–response curves were fitted with the Hill equation.

Fig. 4. NeNaC14 is sensitive to high proton concentrations and NeNaC8 is inhibited by extracellular Ca²⁺.

a Representative current trace of NeNaC14 being activated by increasingly more acidic pH levels (yellow bars). b pH–response curve. c Representative current trace showing opening of NeNaC8 by lowering [Ca²⁺]_e. [Ca²⁺]_e is indicated in µM. d Concentration-response curve. n = 15. e Representative current trace showing inhibition of NeNaC8 by amiloride; NeNaC8 was openend by reducing [Ca²⁺]_e to 100 nM (blue line). f Concentration–response curve. n = 11. g Representative current traces showing inhibition of NeNaC8 by diminazene, dissolved in bath solution containing 100 nM Ca²⁺ (light pink bars). h Representative current traces showing inhibition of NeNaC8 by diminazene, dissolved in bath solution containing 1.8 mM Ca²⁺ (dark pink bars). i Concentration–response curves. n = 14. Error bars represent S.E.M.; Concentration–response curves in b, d, and i were fitted with the Hill equation.

Fig. 5. Diminazene inhibits the time of capturing Artemia nauplii at pH 7.2 and cnidocyst discharge at acidic pH.

a One 3-month-old anemone in a well, ready to capture two swimming Artemia nauplii (red arrows). b the Nematostella polyp captured one of the nauplii with one of its tentacles and drags it to its pharynx. c Box plot with whiskers showing the time in seconds in which polyps capture the first, second, and third nauplius in the absence and in the presence of diminazene (100 µM). d Box plot with whiskers showing the differences in time between the capture of the second and the first, and the third and the second nauplius in the absence and in the presence of diminazene (100 µM). e Pictures of discharged cnidocysts of NvNcol3::mOrange2 positive organisms at two pH values (7.2 and 6.0), with and without prey extract and with and without diminazene. f Bar graph showing the number of discharged cnidocysts (mean ± S.E.) at pH 7.2 with and without prey extract and with and without diminazene. g Bar graph showing the number of discharged cnidocysts (mean ± S.E.) at pH 6.0 with and without prey extract and with and without diminazene. h Gene model of NeNaC2 showing exons and introns; DNA sequences at the bottom show the sequence of the WT and the mutant; the mutant shows a deletion of five nucleotides. The gene region that the sgRNA targeted is indicated in red. i Western blot from NeNaC2 F2 individuals of the three genetic pools showing that the protein was not expressed in NeNaC2^(−/−) animals; GAPDH was used as a control. j Pictures of discharged cnidocysts of the F2 progeny at pH 6.0 and without prey extract from the three genetic pools that were NvNcol3::mOrange2 positive. k Bar graph showing the number of discharged cnidocysts (mean ± S.E.) of the F2 progeny from NeNaC2 of the three genetic pools at pH 6.0 and without prey extract. Unpaired student’s t test from 15 individuals in each treatment for data of c and d. One-way ANOVA of eight organisms per treatment with Bonferroni post-hoc multiple comparisons test for data of f and g. One-way ANOVA from eight individuals per treatment with Tukey post-hoc multiple comparisons test for k. NS, no significant differences, *, ≤0.01; **, ≤0.001, **, ≤0.0001. Scale bars: a, b 1 mm; e 100 µm; h 100 bp; j 100 µm.