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.     

Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid

Bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC gene family of unknown function. Rat BASIC (rBASIC) is inactive at rest. We have recently shown that cholangiocytes, the epithelial cells lining the bile ducts, are the main site of BASIC expression in the liver and identified bile acids, in particular hyo- and chenodeoxycholic acid, as agonists of rBASIC. Moreover, it seems that extracellular divalent cations stabilize the resting state of rBASIC, because removal of extracellular divalent cations opens the channel. In this addendum, we demonstrate that removal of extracellular divalent cations potentiates the activation of rBASIC by bile acids, suggesting an allosteric mechanism. Furthermore, we show that rBASIC is strongly activated by the anticholestatic bile acid ursodeoxycholic acid (UDCA), suggesting that BASIC might mediate part of the therapeutic effects of UDCA.     

The Bile Acid-Sensitive Ion Channel (BASIC) Is Activated by Alterations of Its Membrane Environment

The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.     

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure

The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.     

Mechanotransduction: Touch and Feel at the Molecular Level as Modeled in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The survival of an organism depends on its ability to respond to its environment through its senses. The sense of touch is one of the most vital; still, it is the least understood. In the process of touch sensation, a mechanical stimulus is converted into electrical signals. Groundbreaking electrophysiological experiments in organisms ranging from bacteria to mammals have suggested that this conversion may occur through the activation of ion channels that gate in response to mechanical stimuli. However, the molecular identity of these channels has remained elusive for a very long time. Breakthroughs in our understanding of the cellular and molecular mechanisms of touch sensation have come from the analysis of touch-insensitive mutants in model organisms such as Caenorhabditis elegans and Drosophila melanogaster. This review will focus on the elegant genetic, molecular, imaging, and electrophysiological studies that demonstrate that a channel complex composed of two members of the DEG/ENaC gene family of channel subunits (named for the C. elegans degenerins and the related mammalian epithelial amiloride-sensitive Na channel), MEC-4 and MEC-10, and accessory subunits is gated by mechanical forces in touch-sensing neurons from C. elegans. I also report here electrophysiological and behavioral studies employing knockout mice that have recently shown that mammalian homologues of MEC-4, MEC-10, and accessory subunits are needed for normal mechanosensitivity in mouse, suggesting a conserved function for this channel family across species. The C. elegans genome encodes 28 DEG/ENaC channels: I discuss here the global role of DEG/ENaCs in mechanosensation, reporting findings on the role of other three nematode DEG/ENaCs (UNC-8, DEL-1, and UNC-105) in mechanosensitive and stretch-sensitive behaviors. Finally, this review will discuss findings in which members of another family of ion channels, the Transient Receptor Potential channels family, have been implicated in mechanosensitive behaviors in organisms ranging from C. elegans to mammals.     

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.     

Molecular Modeling of Mechanosensory Ion Channel Structural and Functional Features

The DEG/ENaC (Degenerin/Epithelial Sodium Channel) protein family comprises related ion channel subunits from all metazoans, including humans. Members of this protein family play roles in several important biological processes such as transduction of mechanical stimuli, sodium re-absorption and blood pressure regulation. Several blocks of amino acid sequence are conserved in DEG/ENaC proteins, but structure/function relations in this channel class are poorly understood. Given the considerable experimental limitations associated with the crystallization of integral membrane proteins, knowledge-based modeling is often the only route towards obtaining reliable structural information. To gain insight into the structural characteristics of DEG/ENaC ion channels, we derived three-dimensional models of MEC-4 and UNC-8, based on the available crystal structures of ASIC1 (Acid Sensing Ion Channel 1). MEC-4 and UNC-8 are two DEG/ENaC family members involved in mechanosensation and proprioception respectively, in the nematode Caenorhabditis elegans. We used these models to examine the structural effects of specific mutations that alter channel function in vivo. The trimeric MEC-4 model provides insight into the mechanism by which gain-of-function mutations cause structural alterations that result in increased channel permeability, which trigger cell degeneration. Our analysis provides an introductory framework to further investigate the multimeric organization of the DEG/ENaC ion channel complex.     

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.     

Calreticulin positively regulates the expression and function of epithelial sodium channel

Epithelial sodium channel (ENaC) is a heteromultimeric Na⁺ channel at the apical membrane in the kidney, colon, and lung. Because ENaC plays a crucial role in regulating Na⁺ absorption and extracellular fluid volume, its dysregulation causes severe phenotypes including hypertension, hypokalemia, and airway obstruction. Despite the importance of ENaC, its protein quality control mechanism remains less established. Here we firstly show the role of calreticulin (CRT), a lectin-like molecular chaperone in the endoplasmic reticulum (ER), on the regulation of ENaC. Overexpression and knockdown analyses clearly indicated that CRT positively affects the expression of each ENaC subunit (α, β and γ). CRT overexpression also up-regulated the cell surface expression of α-, β- and γ-ENaC. Moreover, we found that CRT directly interacts with each ENaC subunit. Although CRT knockdown did not affect the de novo synthesis of ENaC subunits, CRT overexpression decreased α-, β- and γ-ENaC expression in the detergent (RIPA)-insoluble fraction, suggesting that CRT enhanced the solubility of ENaC subunits. Consistent with the increased intracellular and cell surface expression of ENaC subunits, increased channel activity of ENaC was also observed upon overexpression of CRT. Our study thus identifies CRT as an ER chaperone that regulates ENaC expression and function.     

DEG/ENaC channels: A touchy superfamily that watches its salt

To the surprise of many, studies of molecular mechanisms of touch transduction and analyses of epithelial Na⁺ transport have converged to define a new class of ion channel subunits. Based on the names of the first two identified subfamilies, the Caenorhabditis elegans degenerins and the vertebrate epithelial amiloride-sensitive Na⁺ channel, this ion channel class is called the DEG/ENaC superfamily. Members of the DEG/ENaC superfamily have been found in nematodes, flies, snails, and vertebrates. Family members share common topology, such that they span the membrane twice and have intracellular N- and C-termini; a large extracellular loop includes a conserved cysteine-rich region. DEG/ENaC channels have been implicated a broad spectrum of cellular functions, including mechanosensation, proprioception, pain sensation, gametogenesis, and epithelial Na⁺ transport. These channels exhibit diverse gating properties, ranging from near constitutive opening to rapid inactivation. We discuss working understanding of DEG/ENaC functions, channel properties, structure/activity correlations and possible evolutionary relationship to other channel classes. BioEssays 21:568–578, 1999. © 1999 John Wiley & Sons, Inc.     

酸敏感离子通道研究进展

组织酸化是生理和病理下常见的现象.神经元可以通过酸敏感的离子通道（ASICs）来感受细胞周围的pH值的降低.ASICs属于NaC/DEG家族的一个成员.目前,已发现了6个ASICs亚基,它们在外周和中枢神经系统中广泛表达,其同聚体和异聚体通道有着各种不同的电生理学特性.ASICs在机体感觉尤其是痛觉中起着至关重要的作用.     

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.     

Accessibility of ENaC extracellular domain central core residues

The epithelial Na⁺ channel (ENaC)/degenerin family has a similar extracellular architecture, where specific regulatory factors interact and alter channel gating behavior. The extracellular palm domain serves as a key link to the channel pore. In this study, we used cysteine-scanning mutagenesis to assess the functional effects of Cys-modifying reagents on palm domain β10 strand residues in mouse ENaC. Of the 13 ENaC α subunit mutants with Cys substitutions examined, only mutants at sites in the proximal region of β10 exhibited changes in channel activity in response to methanethiosulfonate reagents. Additionally, Cys substitutions at three proximal sites of β and γ subunit β10 strands also rendered mutant channels methanethiosulfonate-responsive. Moreover, multiple Cys mutants were activated by low concentrations of thiophilic Cd²⁺. Using the Na⁺ self-inhibition response to assess ENaC gating behavior, we identified four α, two β, and two γ subunit β10 strand mutations that changed the Na⁺ self-inhibition response. Our results suggest that the proximal regions of β10 strands in all three subunits are accessible to small aqueous compounds and Cd²⁺ and have a role in modulating ENaC gating. These results are consistent with a structural model of mouse ENaC that predicts the presence of aqueous tunnels adjacent to the proximal part of β10 and with previously resolved structures of a related family member where palm domain structural transitions were observed with channels in an open or closed state.     

Gating induces a conformational change in the outer vestibule of ENaC

The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma). The channel forms the pathway for Na(+) absorption in the kidney, and mutations cause disorders of Na(+) homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gammaENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na(+) current was increased by mutagenesis or chemical modification of this residue and adjacent residues in alpha, beta, and gammaENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in betaENaC increased the open state probability from 0. 12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na(+) current (gamma(N530K)) was identified in a patient with renal disease.     

COMMD1 regulates the delta epithelial sodium channel (δENaC) through trafficking and ubiquitination

The delta subunit of the epithelial sodium channel (δENaC) is a member of the ENaC/degenerin family of ion channels. δENaC is distinct from the related α-, β- and γENaC subunits, known for their role in sodium homeostasis and blood pressure control, as δENaC is expressed in brain neurons and activated by external protons. COMMD1 (copper metabolism Murr1 domain 1) was previously found to associate with and downregulate δENaC activity. Here, we show that COMMD1 interacts with δENaC through its COMM domain. Co-expression of δENaC with COMMD1 significantly reduced δENaC surface expression, and led to an increase in δENaC ubiquitination. Immunocytochemical and confocal microscopy studies show that COMMD1 promoted localization of δENaC to the early/recycling endosomal pool where the two proteins were localized together. These results suggest that COMMD1 downregulates δENaC activity by reducing δENaC surface expression through promoting internalization of surface δENaC to an intracellular recycling pool, possibly via enhanced ubiquitination.