Acid-sensing ion channels (ASICs) are proton-gated cation channels that are predominantly expressed in the nervous system. ASICs are involved in a number of neurological diseases such as pain, ischemic stroke and multiple sclerosis but limited tools are available to target these channels and provide probes for their physiological functions. Here we report that the anti-protozoal diarylamidines, 4′,6-diamidino-2-phenylindole (DAPI), diminazene, hydroxystilbamidine (HSB) and pentamidine potently inhibit ASIC currents in primary cultured hippocampal neurons with apparent affinities of 2.8 µM, 0.3 µM, 1.5 µM and 38 µM, respectively. These four compounds (100 µM) failed to block ENaC channels expressed in oocytes. Sub-maximal concentrations of diminazene also strongly accelerated desensitization of ASIC currents in hippocampal neurons. Diminazene blocked ASIC1a, -1b -2a, and -3 currents expressed in CHO cells with a rank order of potency 1b > 3 > 2a ≥ 1a. Patchdock computational analysis suggested a binding site of diarylamidines on ASICs. This study indicates diarylamidines constitute a novel class of non-amiloride ASIC blockers and suggests that diarylamidines may be developed as therapeutic agents in treatment of ASIC-involved diseases.

Recently, three ion channel subunits of the degenerin (DEG)/ epithelial Na ؉ channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na ؉ channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamideactivated Na ؉ channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na ؉ selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.The DEG/ENaC 2 gene family contains ion channels that have a high selectivity for Na ϩ ions and a rather high affinity for the diuretic amiloride (apparent IC 50 ϳ 0.1-20 M). In contrast to these conserved pore properties, activating stimuli and physiological functions of DEG/ENaC channels are strikingly diverse (1); family members from Drosophila are involved in the control of locomotion (2), the liquid clearance from the trachea (3), and in the detection of pheromones (4) and salt (5); members from Caenorhabditis elegans form mechanosensitive ion channels (6); FaNaC from snails is a peptide-gated ion channel (7); and the epithelial Na ϩ channel (ENaC) and acid-sensing ion channels family members from mammals mediate Na ϩ reabsorption (8) and are proton-gated ion channels (9), respectively. The functions of many other DEG/ENaC channels are still unknown.Recently, three DEG/ENaC subunits have been cloned from the freshwater polyp Hydra (10). These subunits, HyNaC2-4, were the first DEG/ENaC channels from Cnidaria, which is an ancient phylum, where the first nervous systems are supposed to have evolved. A fourth HyNaC gene,

BackgroundIt is generally the case that fast transmission at neural synapses is mediated by small molecule neurotransmitters. The simple nervous system of the cnidarian Hydra, however, contains a large repertoire of neuropeptides and it has been suggested that neuropeptides are the principal transmitters of Hydra. An ion channel directly gated by Hydra-RFamide neuropeptides has indeed been identified in Hydra – the Hydra Na+ channel (HyNaC) 2/3/5, which is expressed at the oral side of the tentacle base. Hydra-RFamides are more widely expressed, however, being found in neurons of the head and peduncle region. Here, we explore whether further peptide-gated HyNaCs exist, where in the animal they are expressed, and whether they are all gated by Hydra-RFamides.ResultsWe report molecular cloning of seven new HyNaC subunits – HyNaC6 to HyNaC12, all of which are members of the DEG/ENaC gene family. In Xenopus oocytes, these subunits assemble together with the four already known subunits into thirteen different ion channels that are directly gated by Hydra-RFamide neuropeptides with high affinity (up to 40 nM). In situ hybridization suggests that HyNaCs are expressed in epitheliomuscular cells at the oral and the aboral side of the tentacle base and at the peduncle. Moreover, diminazene, an inhibitor of HyNaCs, delayed tentacle movement in live Hydra.ConclusionsOur results show that Hydra has a large variety of peptide-gated ion channels that are activated by a restricted number of related neuropeptides. The existence and expression pattern of these channels, and behavioral effects induced by channel blockers, suggests that Hydra co-opted neuropeptides for fast neuromuscular transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0084-2) contains supplementary material, which is available to authorized users.

Degenerin/epithelial Na+ channels (DEG/ENaCs) are Na+ channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca2+ or have a very low permeability for Ca2+. We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na+ channel (HyNaC), is highly permeable for Ca2+ (PCa/PNa = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca2+-activated Cl− channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on–off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca2+ permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca2+ to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca2+ permeability.

BackgroundIt is generally the case that fast transmission at neural synapses is mediated by small molecule neurotransmitters. The simple nervous system of the cnidarian Hydra, however, contains a large repertoire of neuropeptides and it has been suggested that neuropeptides are the principal transmitters of Hydra. An ion channel directly gated by Hydra-RFamide neuropeptides has indeed been identified in Hydra – the Hydra Na+ channel (HyNaC) 2/3/5, which is expressed at the oral side of the tentacle base. Hydra-RFamides are more widely expressed, however, being found in neurons of the head and peduncle region. Here, we explore whether further peptide-gated HyNaCs exist, where in the animal they are expressed, and whether they are all gated by Hydra-RFamides.ResultsWe report molecular cloning of seven new HyNaC subunits – HyNaC6 to HyNaC12, all of which are members of the DEG/ENaC gene family. In Xenopus oocytes, these subunits assemble together with the four already known subunits into thirteen different ion channels that are directly gated by Hydra-RFamide neuropeptides with high affinity (up to 40 nM). In situ hybridization suggests that HyNaCs are expressed in epitheliomuscular cells at the oral and the aboral side of the tentacle base and at the peduncle. Moreover, diminazene, an inhibitor of HyNaCs, delayed tentacle movement in live Hydra.ConclusionsOur results show that Hydra has a large variety of peptide-gated ion channels that are activated by a restricted number of related neuropeptides. The existence and expression pattern of these channels, and behavioral effects induced by channel blockers, suggests that Hydra co-opted neuropeptides for fast neuromuscular transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0084-2) contains supplementary material, which is available to authorized users.