Physiological insight into the conserved properties of Caenorhabditis elegans acid-sensing degenerin/epithelial sodium channels.

Acid-sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid sensitivity on all 30 subunits of the Caenorhabditis elegans DEG/ENaC family using two-electrode voltage clamp in Xenopus oocytes. We found two groups of acid-sensitive DEG/ENaCs characterised by being either inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified three new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs. KEY POINTS: Acidosis plays many roles in healthy physiology, including synaptic transmission and gut function, but is also a key feature of inflammatory pain, ischaemia and many other conditions. Cells monitor acidosis of their surroundings via pH-sensing channels, including the acid-sensing ion channels (ASICs). These are members of the degenerin/epithelial sodium channel (DEG/ENaC) family, along with, as the name suggests, vertebrate ENaCs and degenerins of the roundworm Caenorhabditis elegans. By screening all 30 C. elegans DEG/ENaCs for pH dependence, we describe, for the first time, three acid-activated members, as well as three additional acid-inhibited channels. We surveyed both groups for sensitivity to amiloride and zinc; like their mammalian counterparts, their currents can be blocked, enhanced or unaffected by these modulators. Likewise, they exhibit diverse ion selectivity. Our findings underline the diversity of acid-sensitive DEG/ENaCs across species and provide a comparative resource for better understanding the molecular basis of their function.

Neuropeptides and degenerin/epithelial Na+ channels: a relationship from mammals to cnidarians.

Ion channels of the degenerin (DEG)/epithelial Na+ channel (ENaC) family serve diverse functions ranging from mechanosensation over Na+ reabsorption to H+ sensing and neurotransmission. However, several diverse DEG/ENaCs interact with neuropeptides; some are directly activated, whereas others are modulated by neuropeptides. Two questions arise: does this interaction have a common structural basis and does it have an ancient origin? Current evidence suggests that RFamide neuropeptides activate the FMRFamide-activated Na+ channels (FaNaCs) of invertebrates via binding to a pocket at the external face of their large extracellular domain. It is likely that RFamides might activate DEG/ENaCs from the freshwater polyp Hydra (the HyNaCs) via binding to a similar pocket, although there is not yet any experimental evidence. In contrast, RFamide neuropeptides modulate acid-sensing ion channels (ASICs) from vertebrates via binding to a central cavity enclosed by ß-sheets of the extracellular domain. Dynorphin opioid peptides, for their part, bind to the acidic pocket of ASICs, which might be evolutionarily related to the peptide binding pocket of FaNaCs, but instead of opening the channels they work as antagonists to stabilize its closed state. Moreover, peptides interacting with DEG/ENaCs from animals of different phyla, although having similar sequences, are evolutionarily unrelated to each other. Collectively, it appears that despite a seemingly similar interaction with similar peptides, the interaction of DEG/ENaCs with neuropeptides has diverse structural bases and many origins.

Is there a role of proinflammatory cytokines on degenerin-mediated cerebrovascular function in preeclampsia?

Preeclampsia (PE) is associated with adverse cerebrovascular effects during and following parturition including stroke, small vessel disease, and vascular dementia. A potential contributing factor to the cerebrovascular dysfunction is the loss of cerebral blood flow (CBF) autoregulation. Autoregulation is the maintenance of CBF to meet local demands with changes in perfusion pressure. When perfusion pressure rises, vasoconstriction of cerebral arteries and arterioles maintains flow and prevents the transfer of higher systemic pressure to downstream microvasculature. In the face of concurrent hypertension, loss of autoregulatory control exposes small delicate microvessels to injury from elevated systemic blood pressure. While placental ischemia is considered the initiating event in the preeclamptic cascade, the factor(s) mediating cerebrovascular dysfunction are poorly understood. Elevated plasma proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-17 (IL-17), are potential mediators of autoregulatory loss. Impaired CBF responses to increases in systemic pressure are attributed to the impaired pressure-induced (myogenic) constriction of small cerebral arteries and arterioles in PE. Myogenic vasoconstriction is initiated by pressure-induced vascular smooth muscle cell (VSMC) stretch. Recent studies from our laboratory group indicate that proinflammatory cytokines impair the myogenic mechanism of CBF autoregulation via inhibition of vascular degenerin proteins, putative mediators of myogenic constriction in VSMCs. This brief review links studies showing the effect of proinflammatory cytokines on degenerin expression and CBF autoregulation to the pathological cerebral consequences of preeclampsia.

Acid-Sensing Ion Channels and Mechanosensation.

Acid-sensing ion channels (ASICs) are mainly proton-gated cation channels that are activated by pH drops and nonproton ligands. They are part of the degenerin/epithelial sodium channel superfamily due to their sodium permeability. Predominantly expressed in the central nervous system, ASICs are involved in synaptic plasticity, learning/memory, and fear conditioning. These channels have also been implicated in multiple disease conditions, including ischemic brain injury, multiple sclerosis, Alzheimer's disease, and drug addiction. Recent research has illustrated the involvement of ASICs in mechanosensation. Mechanosensation is a form of signal transduction in which mechanical forces are converted into neuronal signals. Specific mechanosensitive functions have been elucidated in functional ASIC1a, ASIC1b, ASIC2a, and ASIC3. The implications of mechanosensation in ASICs indicate their subsequent involvement in functions such as maintaining blood pressure, modulating the gastrointestinal function, and bladder micturition, and contributing to nociception. The underlying mechanism of ASIC mechanosensation is the tether-gate model, which uses a gating-spring mechanism to activate ASIC responses. Further understanding of the mechanism of ASICs will help in treatments for ASIC-related pathologies. Along with the well-known chemosensitive functions of ASICs, emerging evidence has revealed that mechanosensitive functions of ASICs are important for maintaining homeostasis and contribute to various disease conditions.

Expression of Exogenous Epithelial Sodium Channel Beta Subunit in the Mouse Middle Cerebral Artery Increases Pressure-Induced Constriction.

BACKGROUND: Pressure-induced constriction (PIC) is inherent to small arteries and arterioles, in which intraluminal pressure-induced vascular smooth muscle cell stretch elicits vasoconstriction. Degenerin (Deg) proteins, such as beta-epithelial Na+ channel (ßENaC), have been studied in the PIC response because they are evolutionarily linked to known mechanosensors. While loss of Deg function phenotypes are plentiful, a gain-of-function phenotype has not been studied. The aim of this study was to determine if expression of exogenous ßENaC in the isolated middle cerebral artery (MCA) enhances the PIC response. METHODS: Isolated MCA segments from female mice (24 weeks, n = 5) were transfected with enhanced green fluorescent protein-ßENaC (EGFP-ßENaC) or with EGFP alone, incubated overnight at 37 °C, then studied in a pressure myograph. RESULTS: Mechanical/morphological properties and vasoconstrictor responses to KCl and phenylephrine were identical in EGFP-ßENaC and EGFP MCAs. In contrast, PIC responses were greater in EGFP-ßENaC segments with ~2-fold greater peak myogenic tone. CONCLUSIONS: These data confirm previous findings that ßENaC is critical in the PIC response. These data provide proof-of-concept that upregulating ßENaC can enhance PIC responses and lay the foundation to test the hypothesis that inflammation-mediated downregulation of ßENaC contributes to cerebrovascular dysfunction.

DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs.

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT­1d, UNC­8d, and MEC­4d formed homomeric channels and that, unlike MEC­4d and UNC­8d, DEGT­1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT­1d and UNC­8d channels. Unexpectedly, MEC­4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.

Calcium ions trigger the exposure of phosphatidylserine on the surface of necrotic cells.

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.

A Na+ leak channel cloned from Trichoplax adhaerens extends extracellular pH and Ca2+ sensing for the DEG/ENaC family close to the base of Metazoa.

Acid-sensitive ion channels belonging to the degenerin/epithelial sodium channel (DEG/ENaC) family activate in response to extracellular protons and are considered unique to deuterostomes. However, sensitivity to pH/protons is more widespread, where, for example, human ENaC Na+ leak channels are potentiated and mouse BASIC and Caenorhabditis elegans ACD-1 Na+ leak channels are blocked by extracellular protons. For many DEG/ENaC channels, extracellular Ca2+ ions modulate gating, and in some cases, the binding of protons and Ca2+ is interdependent. Here, we functionally characterize a DEG/ENaC channel from the early-diverging animal Trichoplax adhaerens, TadNaC6, that conducts Na+-selective leak currents in vitro sensitive to blockade by both extracellular protons and Ca2+ We determine that proton block is enhanced in low external Ca2+ concentration, whereas calcium block is enhanced in low external proton concentration, indicative of competitive binding of these two ligands to extracellular sites of the channel protein. TadNaC6 lacks most determinant residues for proton and Ca2+ sensitivity in other DEG/ENaC channels, and a mutation of one conserved residue (S353A) associated with Ca2+ block in rodent BASIC channels instead affected proton sensitivity, all indicative of independent evolution of H+ and Ca2+ sensitivity. Strikingly, TadNaC6 was potently activated by the general DEG/ENaC channel blocker amiloride, a rare feature only reported for the acid-activated channel ASIC3. The sequence and structural divergence of TadNaC6, coupled with its noncanonical functional features, provide unique opportunities for probing the proton, Ca2+, and amiloride regulation of DEG/ENaC channels and insight into the possible core-gating features of ancestral ion channels.

The synaptic action of Degenerin/Epithelial sodium channels.

Degenerin/Epithelial Sodium Channels (DEG/ENaCs) are a large family of animal-specific non-voltage gated ion channels, with enriched expression in neuronal and epithelial tissues. While neuronal DEG/ENaCs were originally characterized as sensory receptor channels, recent studies indicate that several DEG/ENaC family members are also expressed throughout the central nervous system. Human genome-wide association studies have linked DEG/ENaC-coding genes with several neurologic and psychiatric disorders, including epilepsy and panic disorder. In addition, studies in rodent models further indicate that DEG/ENaC activity in the brain contributes to many behaviors, including those related to anxiety and long-term memory. Although the exact neurophysiological functions of DEG/ENaCs remain mostly unknown, several key studies now suggest that multiple family members might exert their neuronal function via the direct modulation of synaptic processes. Here, we review and discuss recent findings on the synaptic functions of DEG/ENaCs in both vertebrate and invertebrate species, and propose models for their possible roles in synaptic physiology.

The degenerin region of the human bile acid-sensitive ion channel (BASIC) is involved in channel inhibition by calcium and activation by bile acids.

The bile acid-sensitive ion channel (BASIC) is a member of the ENaC/degenerin family of ion channels. It is activated by bile acids and inhibited by extracellular Ca2+. The aim of this study was to explore the molecular mechanisms mediating these effects. The modulation of BASIC function by extracellular Ca2+ and tauro-deoxycholic acid (t-DCA) was studied in Xenopus laevis oocytes heterologously expressing human BASIC using the two-electrode voltage-clamp and outside-out patch-clamp techniques. Substitution of aspartate D444 to alanine or cysteine in the degenerin region of BASIC, a region known to be critically involved in channel gating, resulted in a substantial reduction of BASIC Ca2+ sensitivity. Moreover, mutating D444 or the neighboring alanine (A443) to cysteine significantly reduced the t-DCA-mediated BASIC stimulation. A combined molecular docking/simulation approach demonstrated that t-DCA may temporarily form hydrogen bonds with several amino acid residues including D444 in the outer vestibule of the BASIC pore or in the inter-subunit space. By these interactions, t-DCA may stabilize the open state of the channel. Indeed, single-channel recordings provided evidence that t-DCA activates BASIC by stabilizing the open state of the channel, whereas extracellular Ca2+ inhibits BASIC by stabilizing its closed state. In conclusion, our results highlight the potential role of the degenerin region as a critical regulatory site involved in the functional interaction of Ca2+ and t-DCA with BASIC.

Modulation of DEG/ENaCs by Amphiphiles Suggests Sensitivity to Membrane Alterations.

The bile acid-sensitive ion channel is activated by amphiphilic substances such as bile acids or artificial detergents via membrane alterations; however, the mechanism of membrane sensitivity of the bile acid-sensitive ion channel is not known. It has also not been systematically investigated whether other members of the degenerin/epithelial Na+ channel (DEG/ENaC) gene family are affected by amphiphilic compounds. Here, we show that DEG/ENaCs ASIC1a, ASIC3, ENaC, and the purinergic receptor P2X2 are modulated by a large number of different, structurally unrelated amphiphilic substances, namely the detergents N-lauroylsarcosine, Triton X-100, and ß-octylglucoside; the fenamate flufenamic acid; the antipsychotic drug chlorpromazine; the natural phenol resveratrol; the chili pepper compound capsaicin; the loop diuretic furosemide; and the antiarrythmic agent verapamil. We determined the modification of membrane properties using large-angle x-ray diffraction experiments on model lipid bilayers, revealing that the amphiphilic compounds are positioned in a characteristic fashion either in the lipid tail group region or in the lipid head group region, demonstrating that they perturbed the membrane structure. Collectively, our results show that DEG/ENaCs and structurally related P2X receptors are modulated by diverse amphiphilic molecules. Furthermore, they suggest alterations of membrane properties by amphiphilic compounds as a mechanism contributing to modulation.

The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide.

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.

The Drosophila Postsynaptic DEG/ENaC Channel ppk29 Contributes to Excitatory Neurotransmission.

The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.

Mechanosensory molecules and circuits in C. elegans.

Mechanosensory neurons, whose activity is controlled by mechanical force, underlie the senses of touch, hearing, and proprioception, yet despite their importance, the molecular basis of mechanotransduction is poorly understood. Genetic studies in Caenorhabditis elegans have provided a useful approach for identifying potential components of mechanotransduction complexes that might be conserved in more complex organisms. This review describes the mechanosensory systems of C. elegans, including the sensory neurons and circuitry involved in body touch, nose touch, and proprioception. In addition, the roles of genes encoding known and potential mechanosensory receptors, including members of the broadly conserved transient receptor potential (TRP) and degerin/epithelial Na(+) channel (DEG/ENaC) channel families, are discussed.

Identification of Ppk26, a DEG/ENaC Channel Functioning with Ppk1 in a Mutually Dependent Manner to Guide Locomotion Behavior in Drosophila.

A major gap in our understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, we identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1, another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception.

Balboa binds to pickpocket in vivo and is required for mechanical nociception in Drosophila larvae.

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In this study, in a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, we identify a gene that we name balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led us to hypothesize that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, we directly detect in vivo physical interactions among PPK and Balboa subunits. Combined, our results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.

The comprehensive analysis of DEG/ENaC subunits in Hydra reveals a large variety of peptide-gated channels, potentially involved in neuromuscular transmission.

BACKGROUND: It 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Pharmacological and electrophysiological characterization of the human bile acid-sensitive ion channel (hBASIC).

The human bile acid-sensitive ion channel (hBASIC) is a cation channel of the degenerin/epithelial Na(+) channel gene family that is expressed in the intestinal tract and can be activated by bile acids. Here, we show that in addition to its sensitivity for bile acids, hBASIC shares further key features with its rat ortholog: it is blocked by extracellular divalent cations, is inhibited by micromolar concentrations of the diarylamidine diminazene, and activated by millimolar concentrations of flufenamic acid. Furthermore, we demonstrate that two major bile acids present in human bile, chenodeoxycholic acid and deoxycholic acid, activate hBASIC in a synergistic manner. In addition, we determined the single-channel properties of hBASIC in outside-out patch clamp recordings, revealing a single-channel conductance of about 11 pS and a high Na(+) selectivity. Deoxycholic acid activates hBASIC in patch clamp recordings mainly by reducing the single-channel closed time. In summary, we provide a thorough functional characterization of hBASIC.

Molecular determinants of desensitization in an ENaC/degenerin channel.

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Šabove the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

ENaC in the brain--future perspectives and pharmacological implications.

The epithelial sodium channel/degenerin (ENaC/deg) family of ion channels is formed by a large number of genes with variable tissue expression patterns and physiological roles. ENaC is a non-voltage gated, constitutively active channel highly selective for sodium. ENaC is formed by three homologous subunits, α, ß and γ, and a fourth subunit (δ) has been found in human and monkeys that can substitute α to form functional channels. The best-characterized role of ENaC is to serve as a rate-limiting step in transepithelial sodium reabsorption in the distal part of the kidney tubule and other tight epithelia. However, ENaC subunits are also found in the peripheral and central nervous system, where their functional roles are only beginning to be understood. In this review, we mainly focus on the putative pathophysiological roles of ENaC channels in the central nervous system and their potential value as drug targets in neurodegenerative disorders and the central control of blood pressure.