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	Figure 1. Consensus tree for the DEG/ENaC family. The phylogenetic tree was generated by Bayesian analysis with the program MrBayes 3.2 (see Methods). The numbers at nodes indicate the posterior probabilities computed by MrBayes for the respective node. Scale bar indicates amino acid exchanges per site. Colors indicate the following phyla: blue, Cnidaria; red, Chordata; green, Arthropoda; brown, Nematoda; pink, Mollusca. Abbrevations of species names are as follows: c, chicken (Gallus gallus); h, human (Homo sapiens); Ha, Helix aspersa; Ht, Helisoma trivolvis; l, lamprey (Lampetra fluviatilis), Ls, Lymnaea stagnalis; m, mouse (Mus musculus); r, rat (Rattus norvegicus). Other proteins are from C. elegans, Drosophila melanogaster or Hydra magnipapillata. Accession numbers are as follows: acid-sensitive degenerin (ACD)-1 [GenBank:NM_058894], ACD-2 [GenBank:NM_058892], ACD-4 [GenBank:NM_072829], ACD-5 [GenBank:NM_058795], cASIC1 [EMBL:AY956393], cASIC2 [GenBank:NM_001040467], hASIC1a [EMBL:U78180], hASIC2a [EMBL:U50352], hASIC2b [GenBank:NM_18337], hASIC3 [EMBL:AF095897], hASIC4 [EMBL:AJ271643], lASIC1 [EMBL:AAY28983], rASIC1a [EMBL:U94403], rASIC1b [EMBL:AJ309926], rASIC2a [EMBL:U53211], rASIC2b [EMBL:AB049451], rASIC3 [EMBL:AF013598], rASIC4 [EMBL:AJ271642], hBASIC [EMBL:AJ252011], mBASIC [EMBL:Y19035], rBASIC [EMBL:Y19034], DEG-1 NM_076910; degenerin-like (DEL)-1 [EMBL:U76403], DEL-4 [GenBank:NM_059829], DEL-7 [GenBank:NM_068875], DEL-9 [GenBank:NM_076221], DEL-10 [GenBank:NM-062901], αhENaC [EMBL:X76180], βhENaC [EMBL:X87159], γhENaC [EMBL:X87160], δhENaC [EMBL:U38254], αrENaC [EMBL:X70521], βrENaC [EMBL:X77932], γrENaC [EMBL:X77933], HtFaNaC [EMBL:AF254118], LsFaNaC [EMBL:AF335548], HaFaNaC [EMBL:X92113], fluoride-resistant (FLR)-1 [EMBL:AB012617], HyNaC2 [EMBL:AM393878], HyNaC3 [EMBL:AM393880], HyNaC4 [EMBL:AM393881], HyNaC5 [EMBL:FN257513], HyNaC6 [EMBL:HG422725], HyNaC7 [EMBL:HG422726], HyNaC8 [EMBL:HG422727], HyNaC9 [EMBL:HG422728], HyNaC10 [EMBL:HG422729], HyNaC11 [EMBL:HG422730], HyNaC12 [EMBL:HG422731], mechanosensitive (MEC)-4 [EMBL:X58982], MEC-10 [EMBL:L25312], pickpocket (PPK) [EMBL:Y16225], PPK4 [GenBank:NM_206137], PPK6 [GenBank:NM_137617], PPK7 [GenBank:NM_135172], PPK10 [GenBank:NM_001038805], PPK11 [GenBank:NM_001038798], PPK12 [GenBank:NM_137828], PPK13 [GenBank:NM_001014495], PPK16 [GenBank:NM_001038797], PPK17 [GenBank:NM_135965], PPK19 [EMBL:AY226547], PPK20 [GenBank:NM_143448], PPK21 [GenBank:NM_143447], PPK23 [GenBank:NM_001014749], PPK24 [GenBank:NM_143603], PPK25 [GenBank:NM_206044], PPK26 [GenBank:NM_139868], PPK27 [GenBank:NM_139569], PPK28 [GenBank:NM_001014748], ripped pocket (RPK) [EMBL:Y12640], uncoordinated (UNC)-8 [EMBL:U76402], UNC-105 [GenBank:NM_063301].
	Figure 2. Sequence alignment of HyNaC2 – HyNaC12 with ASIC1a and BASIC. HyNaCs share conserved motifs and structures that are typical for DEG/ENaCs. Conserved amino acids are highlighted as white letters on black background. Bars indicate the putative position of TMDs and circles conserved cysteines in the ECD. The selectivity filter in TMD2 is indicated by an ellipse and the conserved N-terminal HG motif by an open bar. Accession numbers can be found in the legend to Figure 1. ASIC, acid-sensing ion channel; BASIC, bile-acid sensitive ion channel; ECD, extracellular domain; HyNaC, Hydra Na+ channel; TMDs, transmembrane domains.
	Figure 3. Rules of subunit assembly. Left, the table shows possible trimeric combinations of HyNaCs. HyNaC2 has to be present for a peptide-gated HyNaC. Subunits in the top row and the left row belong to the two subgroups defined by phylogenetic relation. A functional channel activated by Hydra-RFamides is represented by ‘ + ‘, a non-functional channel by ‘-‘. Activation by Ca2+ removal but insensitivity to Hydra-RFamides is indicated by ‘(+)’. Subunit combinations that are co-expressed in Hydra as revealed by ISH are shown on colored background. Orange represents expression at the oral side of the tentacle base, blue at the aboral side and green at the peduncle. Right, scheme illustrating expression sites of HyNaCs. HyNaC, Hydra Na+ channel; ISH, in situ hybridization.
	Figure 4. HyNaCs are insensitive to Hydra-RFamides III to V. Representative current trace showing activation of HyNaCs by the removal of extracellular divalent cations or by 5 μM Hydra-RFamide I. In contrast, 5 μM Hydra-RFamide III, IV or V did not elicit a current for any functional HyNaC heterotrimer; a current trace from HyNaC2/11/5 is shown as an example. Dashed line represents the zero current level. Amino acid sequences of Hydra-RFamides I to V are shown at the bottom. HyNaC, Hydra Na+ channel.
	Figure 5. hynacs are expressed at the base of the tentacles or at the peduncle. Whole mount in situ hybridizations revealed a diverse expression of different HyNaCs. hynac2 to hynac6 and hynac11 are expressed at the base of the tentacles, while hynac7 and hynac10 are expressed at the peduncle. hynac8 and hynac9 are expressed ubiquitously along the whole body column. Note that hynac2 is faintly expressed over the whole body column including the peduncle. Expression of hynac12 could not be detected by in situ hybridization.
	Figure 6. hynacs are differentially expressed at the base of the tentacles or at the peduncle. Magnifications of the in situ hybridizations for hynac6, hynac7 and hynac9 to hynac11 from Figure 5, demonstrating more precisely their expression patterns at the tentacle base and the peduncle.
	Figure 7. HyNaCs are activated by Hydra-RFamide I. A) Representative current traces showing concentration-dependent activation of HyNaC2/3/6 and HyNaC2/9/7 by Hydra-RFamide I when oocytes had been injected with EGTA. Dashed lines represent the zero current level. B) When oocytes had not been injected with EGTA, Hydra-RFamide I elicited biphasic currents; an oocyte expressing HyNaC2/9/7 is shown as an example. C) Concentration-response curves for HyNaCs and Hydra-RFamide I. Currents were normalized to the currents at the highest agonist concentration, which had amplitudes of 9.2 ± 1.9 μA (n = 9; 2/3/5), 2.6 ± 0.4 μA (n = 12; 2/11/5), 15.7 ± 3.2 μA (n = 8; 2/3/6), 10.1 ± 1.7 μA (n = 12; 2/4/6), 19.6 ± 1.8 μA (n = 10; 2/3/7), 7.2 ± 0.9 μA (n = 10; 2/9/7) and 10.6 ± 1.8 μA (n = 8; 2/10/7), respectively. Lines represent fits to the Hill equation. D) I/V curves for putative physiological HyNaCs, revealing slightly positive reversal potentials. Voltage ramps were run from −100 mV to +30 mV in two seconds. Background currents had been subtracted by voltage ramps in the absence of agonist. Currents were normalized to the current at −100 mV. EGTA, ethylene glycol tetraacetic acid; HyNaC, Hydra Na+ channel.
	Figure 8. HyNaCs are activated by Hydra-RFamide II. A) Representative current traces showing concentration-dependent activation of HyNaC2/4/6 and HyNaC2/11/5 by Hydra-RFamide II; oocytes had been injected with EGTA. Dashed lines represent the zero current level. B) Concentration-response curves for HyNaCs and Hydra-RFamide II. Currents were normalized to the currents at highest agonist concentration, which had amplitudes of 5.3 ± 1.0 μA (n = 14; 2/3/5), 2.2 ± 0.4 μA (n = 7; 2/11/5), 9.2 ± 1.7 μA (n = 8; 2/3/6), 4.7 ± 0.8 μA (n = 12; 2/4/6), 11.8 ± 4.0 μA (n = 8; 2/3/7), 3.6 ± 1.3 μA (n = 8; 2/9/7) and 1.1 ± 0.4 μA (n = 7; 2/10/7), respectively. Lines represent fits to the Hill equation. EGTA, ethylene glycol tetraacetic acid; HyNaC, Hydra Na+ channel.
	Figure 9. Change in extracellular Ca 2+ concentration shifts the reversal potential of HyNaCs. A) Xenopus oocytes expressing HyNaC2/3/7 were activated by 100 nM Hydra-RFamide I, corresponding to its EC50. The conductive cation in the extracellular solution was either 1 mM Ca2+, or 10 mM Ca2+, or 140 mM Na+. Reversal potentials were determined by voltage ramps from −100 mV to +30 mV in two seconds. Background currents had been subtracted by voltage ramps in the absence of agonist. B) Diagram showing the shift of the reversal potentials for different HyNaC combinations, when the extracellular solution contained 1 or 10 mM Ca2+ as the only conductive ion. HyNaCs had been activated by a concentration of Hydra-RFamide I that corresponds to the individual EC50. HyNaCs, Hydra Na+ channels.
	Figure 10. Diminazene is a potent inhibitor of HyNaCs. A) Current trace illustrating the concentration-dependent inhibition of HyNaC2/9/7 by diminazene. Block by 100 μM amiloride was used to compare the affinity for diminazene and amiloride. Note that current amplitude transiently increased after washout of the blockers. Dashed lines represent the zero current level. B) Concentration-response curves showing the concentration-dependent inhibition of HyNaCs by diminazene. HyNaCs, Hydra Na+ channels.
	Figure 11. Diminazene inhibits the feeding reaction of Hydra magnipapillata . Adult animals were held in plain medium or in medium containing 100, 200 or 300 μM diminazene (Dimi). The feeding response was induced by application of 10 μM glutathione (GSH). The number of animals showing complete tentacle curling was recorded every 30 seconds. *, P <0.05; **, P <0.01; ***, P <0.001.

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