The bile acid-sensitive ion channel (BASIC), the ignored cousin of ASICs and ENaC

The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na⁺ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.     

The bile acid-sensitive ion channel (BASIC), the ignored cousin of ASICs and ENaC

The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na⁺ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.     

Molecular and functional identification of sodium channels in K562 cells

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various nonexcitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na⁺ channels (DEG/ENaC) can be considered as possible molecular correlates. By using RT-PCR, expression of ??-, ??-, and ??-hENaC subunits in the K562 cells was detected. Various modes of the patch-clamp method were used to examine functional properties of sodium channels??specifically, to test the effect of amiloride on single channel and integral currents. The biophysical characteristics of the NVSG channels were close to those of ENaC; the channels have unitary conductance of 12 pS (145 mM Na⁺) and were impermeable to divalent cations (Ca²⁺ and Mg²⁺). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in the K562 cells belong to the ENaC family.     

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na⁺ channels facilitate the transport of Na⁺ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na⁺ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.Many ion channels are silent at rest and are activated in response to a variety of factors, including membrane potential, external ligands, and intracellular signaling processes. The ENaC² has evolved as a channel that is thought to reside primarily in an active state, facilitating the bulk movement of Na⁺ out of renal tubular or airway lumens. The regulated insertion and retrieval of channels at the plasma membrane have important roles in modulating ENaC-dependent Na⁺ transport (1). A number of factors also have a role in regulating ENaC activity via changes in channel P_o or gating. In this regard, it has become increasingly apparent that proteolysis of ENaC subunits has a key role in this process (2). This minireview addresses several questions regarding the role of ENaC subunit proteolysis in regulating channel gating. (i) Where are ENaC subunits cleaved? (ii) Which proteases mediate ENaC cleavage? (iii) Why are channels activated by proteolysis? (iv) Is proteolysis responsible, in part, for the highly variable channel P_o that has been noted for ENaC? (v) Why have ENaCs evolved as channels that require proteolysis for activation?     

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

The epithelial Na⁺ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na⁺, Cl⁻, protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na⁺ concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na⁺ binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na⁺. Mutations at selected sites altered the cation inhibitory preference to favor Li⁺ or K⁺ rather than Na⁺. Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na⁺. Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.     

Regulation of the epithelial sodium channels (ENaC) by small G proteins and phosphatidylinositides

The epithelial Na⁺ channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. ENaC activity in these epithelia is limiting for sodium reabsorption. Abnormalities in ENaC function have been linked to disorders of total body Na⁺ homeostasis, blood volume, blood pressure, and lung fluid balance. Recently, ion channels were recognized as physiologically important effectors of small GTP-binding proteins and phosphatidylinositides. We review here recent findings relevant to regulation of ENaC by small G proteins and phosphatidylinositides.     

Frog atrial natriuretic peptide and cGMP activate amiloride-sensitive Na<Superscript>+</Superscript> channels in urinary bladder cells of Japanese tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

In our previous study, it was suggested that ANP and cGMP may increase Na⁺ absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na⁺ transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na⁺ channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 ± 0.2 pS, long open and closed times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including 10⁻⁶ M amiloride. These characteristics were similar to those of amiloride-sensitive Na⁺ channels (ENaC). Addition of 10⁻⁹ M ANP activated and significantly increased the ENaC activity from 0.58 ± 0.09 to 1.47 ± 0.34. On the other hand, mean amplitudes and conductance of single channel did not change significantly after the addition of ANP. Addition of 10⁻⁵ M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 ± 0.10 to 2.00 ± 0.33. The addition of ANP failed to activate the ENaC in the presence of 10⁻⁶ M amiloride. These results suggested that ANP and cGMP activate Na⁺ transport via ENaC in the epithelial cells of frog urinary bladder.     

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl⁻ concentrations in the kidney and in other epithelia. We tested whether Cl⁻ alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl⁻ with SO₄²⁻, H₂PO₄⁻, or SCN⁻ produced a large increase in ENaC current, indicating that extracellular Cl⁻ inhibits ENaC. Extracellular Cl⁻ also inhibited ENaC in Na⁺-transporting epithelia. The anion selectivity sequence was SCN⁻ < SO₄²⁻ < H₂PO₄⁻ < F⁻ < I⁻ < Cl⁻ < Br⁻. Crystallization of ASIC1a revealed a Cl⁻ binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (α_H418A and β_R388A) disrupted the response to Cl⁻, suggesting that Cl⁻ might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl⁻. The response to Cl⁻ was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl⁻. Cl⁻ regulated ENaC activity in part through enhanced Na⁺ self-inhibition, a process by which extracellular Na⁺ inhibits ENaC. Together, the data indicate that extracellular Cl⁻ regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl⁻ might modulate epithelial Na⁺ absorption.The epithelial Na⁺ channel ENaC² is a heterotrimer of homologous α, β, and γ subunits (1, 2). ENaC functions as a pathway for Na⁺ absorption across epithelial cells in the kidney collecting duct, lung, distal colon, and sweat duct (reviewed in Refs. 3 and 4). Na⁺ transport is critical for the maintenance of Na⁺ homeostasis and for the control of the composition and quantity of the fluid on the apical membrane of these epithelia. ENaC mutations and defects in its regulation cause inherited forms of hypertension and hypotension (5) and may contribute to the pathogenesis of lung disease in cystic fibrosis (6).ENaC is a member of the DEG/ENaC family of ion channels. A common structural feature of these channels is a large extracellular domain that plays a critical role in channel gating. For example, in ASICs, the extracellular domain functions as a receptor for protons, which transiently activate the channel by titrating residues that form an acidic pocket (7). FaNaCh is a ligand-gated family member in Helix aspersa, activated by the peptide FMRFamide (8). In Caenorhabditis elegans MEC family members, the extracellular domain is thought to respond to mechanical signals (9).ENaC differs from other family members because it is constitutively active in the absence of a ligand/stimulus. However, a convergence of data indicate that ENaC gating is modulated by a variety of molecules that bind to or modify its extracellular domains, including proteases (10–12), Na⁺ (13–15), protons (16), and the divalent cations Zn²⁺ and Ni²⁺ (17, 18). These findings suggest that the ENaC extracellular domain might regulate epithelial Na⁺ transport by sensing and integrating diverse signals in the extracellular environment.In the current study, we tested the hypothesis that ENaC activity is regulated by changes in the extracellular Cl⁻ concentration. Several observations suggested that Cl⁻ might be a strong candidate to regulate the channel. First, transport of Na⁺ and Cl⁻ are often coupled to maintain electroneutrality. Second, ENaC is exposed to large changes in extracellular Cl⁻ concentration. For example, in the kidney collecting duct, the urine Cl⁻ concentration varies widely (19). As the predominant anion, its concentration parallels that of Na⁺ in most clinical states. However, under conditions of metabolic alkalosis and metabolic acidosis, the Na⁺ and Cl⁻ concentrations can become dissociated as a result of increased urinary bicarbonate (alkalosis) or ammonium (acidosis) (19). Thus, ENaC is well positioned to respond to changes in Cl⁻ concentration. Third, crystallization of ASIC1a revealed a binding site for a Cl⁻ ion at the base of the thumb domain (7). The Cl⁻ is coordinated by Arg-310 and Glu-314 from one subunit and Lys-212 from an adjacent subunit. Although the functional role of Cl⁻ binding to ASIC1a is unknown, it supports the hypothesis that extracellular Cl⁻ might regulate the activity of DEG/ENaC ion channels.     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle''s syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle''s mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

High Ca2+ permeability of a peptide-gated DEG/ENaC from Hydra

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⁺ (P_Ca/P_Na = 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.     

Control of Epithelial Ion Transport by Cl<Superscript>−</Superscript> and PDZ Proteins

Inhibition of epithelial Na⁺ channels (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated previously. Recent studies suggested a role of cytosolic Cl⁻ for the interaction of CFTR with ENaC, when studied in Xenopus oocytes. In the present study we demonstrate that the Na⁺/H⁺-exchanger regulator factor (NHERF) controls expression of CFTR in mouse collecting duct cells. Inhibition of NHERF largely attenuates CFTR expression, which is paralleled by enhanced Ca²⁺-dependent Cl⁻ secretion and augmented Na⁺ absorption by the ENaC. It is further demonstrated that epithelial Na⁺ absorption and ENaC are inhibited by cytosolic Cl⁻ and that stimulation by secretagogues enhances the intracellular Cl⁻ concentration. Thus, the data provide a clue to the question, how epithelial cells can operate as both absorptive and secretory units: Increase in intracellular Cl⁻ during activation of secretion will inhibit ENaC and switch epithelial transport from salt absorption to Cl⁻ secretion.This revised version was published online in August 2005 with a corrected cover date.     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

A glial DEG/ENaC channel functions with neuronal channel DEG-1 to mediate specific sensory functions in C. elegans

Mammalian neuronal DEG/ENaC channels known as ASICs (acid-sensing ion channels) mediate sensory perception and memory formation. ASICS are closed at rest and are gated by protons. Members of the DEG/ENaC family expressed in epithelial tissues are called ENaCs and mediate Na(+) transport across epithelia. ENaCs exhibit constitutive activity and strict Na(+) selectivity. We report here the analysis of the first DEG/ENaC in Caenorhabditis elegans with functional features of ENaCs that is involved in sensory perception. ACD-1 (acid-sensitive channel, degenerin-like) is constitutively open and impermeable to Ca(2+), yet it is required with neuronal DEG/ENaC channel DEG-1 for acid avoidance and chemotaxis to the amino acid lysine. Surprisingly, we document that ACD-1 is required in glia rather than neurons to orchestrate sensory perception. We also report that ACD-1 is inhibited by extracellular and intracellular acidification and, based on the analysis of an acid-hypersensitive ACD-1 mutant, we propose a mechanism of action of ACD-1 in sensory responses based on its sensitivity to protons. Our findings suggest that channels with ACD-1 features may be expressed in mammalian glia and have important functions in controlling neuronal function.     

Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity

FMRFamide (Phe-Met-Arg-Phe-amide, FMRFa) and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFa activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFa potency against one of its native targets, the excitatory FMRFa-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of neuropeptide activity, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFa, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching degenerin (DEG)/epithelial sodium channel (ENaC) superfamily, incorporating mutagenesis and experimental dissection of channel function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands but enhanced by FMRFa, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFa neuropeptide activity at FaNaC receptor channels and illuminates the evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.     

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 β-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 epithelial sodium channel: from molecule to disease

Genetic analysis has demonstrated that Na absorption in the aldosterone-sensitive distal nephron (ASDN) critically determines extracellular blood volume and blood pressure variations. The epithelial sodium channel (ENaC) represents the main transport pathway for Na⁺ absorption in the ASDN, in particular in the connecting tubule (CNT), which shows the highest capacity for ENaC-mediated Na⁺ absorption. Gain-of-function mutations of ENaC causing hypertension target an intracellular proline-rich sequence involved in the control of ENaC activity at the cell surface. In animal models, these ENaC mutations exacerbate Na⁺ transport in response to aldosterone, an effect that likely plays an important role in the development of volume expansion and hypertension. Recent studies of the functional consequences of mutations in genes controlling Na⁺ absorption in the ASDN provide a new understanding of the molecular and cellular mechanisms underlying the pathogenesis of salt-sensitive hypertension.