Amiloride-sensitive Ion Channels Research Articles

Functional and Pharmacological Characterization of C. elegans DEG/ENaC/ASIC Channels

Activating or blocking ion channels is a central goal of drug discovery. However, ion channel families have multiple variants which differ in function. Additionally, channel subunits assemble to form multimeric complexes with varying biophysical properties and drug sensitivities. Degenerin (DEG)/epithelial sodium channel (ENaC)/acid-sensing ion channel (ASIC) proteins arebelieved to assemble as homomeric or heteromeric trimers into non-voltage gated, sodium-selective, and amiloride sensitive ion channels. Here, we analyzed a subset of the 30 DEG/ENaC/ASIC proteins encoded by the C. elegans genome in order to test the hypothesis that individual family members have diverse properties. Specifically, we expressed five C. elegans isoforms in Xenopus laevis oocytes (DEGT-1d, DEL-1d, UNC-8d, MEC-10d and MEC-4d) and measured current amplitude, selectivity among monovalent cations, sensitivity to amiloride and its analogs, and sensitivity to nonsteroidal anti-inflammatory drugs (NSAIDs). Overall, we find substantial variations in the pharmacological footprints of the two drug families we studied (amiloride, NSAIDs) and among the DEG/ENaC/ASIC proteins. DEGT-1d is the first channel in this family demonstrated to be equally permeable to sodium and potassium, and has no response to amiloride and its analogs. We identified at least one NSAID that blocked DEGT-1d and UNC-8d currents. Surprisingly, MEC4d currents were potentiated by five NSAIDs. A mutation homologous to ASIC1a[K422], MEC4[E704K], decreased the sensitivity to NSAIDs. The molecular origin of this potentiation will be the focus of future studies, along with identifying which structural elements are responsible for inhibition and potentiation. This work will help to advance drug design for this family and enhance our understanding of how NSAIDs exert their function. Collectively, these data establish that DEG/ENaC/ASIC ion channel proteins vary in their ion permeability and drug sensitivity. Work supported by DFG (SF), SSRP (ID), R01NS047715, R35NS129879 (MBG)

The Thiazide-Sensitive Co-Transporter Promotes the Development of Sodium Retention in Mice with Diet-Induced Obesity

Background/Aims: Intravascular volume expansion due to sodium retention is involved in the pathogenesis of obesity-related hypertension. Institution of high fat diet (HFD) feeding leads to an initial state of positive sodium balance due to enhanced tubular reabsorption of sodium, but which tubular sodium transporters are responsible for this remains undefined. Methods: C57/Bl6 mice were fed control or HFD for 3 weeks. Blood pressures were recorded by tail cuff method. Sodium transporter expression and phosphorylation were determined by Western blotting. In vivo activity of NCC was determined using natriuretic responses to hydrochlorothiazide. Expression of NCC mRNA was determined using qPCR. Results: At 3 weeks HFD mice had significant weight gains compared to control mice, but blood pressures were not yet elevated. There were no changes in expression or phosphorylation of the bumetanide-sensitive cotransporter, NKCC2, or in expression of subunits of the amiloride-sensitive ion channel, ENaC. However, there were significant increases in mRNA and protein expression of the thiazide-sensitive co-transporter, NCC, in kidneys from HFD mice. Consistent with this, HFD mice had increased in vivo activity of NCC. Conclusions: Increased expression of NCC promotes the sodium loading response to institution of HFD feeding before onset of hypertension.

Regulation of the delta and alpha epithelial sodium channel (ENaC) by ubiquitination and Nedd8

The δ epithelial sodium channel (δENaC) is a proton-activated, sodium-selective, amiloride-sensitive ion channel in the ENaC/degenerin family of ion channels involved in blood pressure regulation and mechanosensation. Other ENaC family members are subject to ubiquitin modification leading to internalization from the cell surface, and degradation of the channel. Here, we show that δENaC is also modified by ubiquitin on three intracellular lysine residues. Absence of these lysines abolished ubiquitin modification of δENaC and increased cell surface levels of δENaC. Although the HECT-domain ubiquitin ligase Nedd4-2 reduced amiloride-sensitive current generated by δβγENaC-containing channels, δENaC does not contain a binding site for Nedd4-2; therefore, this effect is probably mediated by the βγENaC subunits. Nedd8, a ubiquitin-like protein that regulates RING-domain E3 ubiquitin ligases, promoted δENaC ubiquitination, decreased both the intracellular and cell surface δENaC populations, and decreased δβγENaC amiloride-sensitive short circuit current (Isc -amiloride) in a mammalian epithelium. Nedd8 also promoted α- and γENaC ubiquitination, decreased the cell surface pools, and decreased αβγENaC Isc -amiloride. Conversely, XIAP, a single subunit RING E3 ligase, decreased ubiquitinated δENaC, increased the δENaC cell surface pool and increased δβγENaC Isc -amiloride. Therefore δ- and α - βγENaC channel function may be influenced by RING-domain E3 ubiquitin ligases.

Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences

Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target.

Heteromeric Assembly of Acid-sensitive Ion Channel and Epithelial Sodium Channel Subunits

Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.

994: Amiloride-Sensitive Ion Channels in Urinary Bladder Epithelium Involved in Mechanosensory Transduction by Modulating Stretch-Evoked ATP Release

You have accessJournal of UrologyModerated Poster 30, Monday, May 21, 2007, 1:00 - 3:00 pm1 Apr 2007994: Amiloride-Sensitive Ion Channels in Urinary Bladder Epithelium Involved in Mechanosensory Transduction by Modulating Stretch-Evoked ATP Release Shuqi Du, Isao Araki, Yuki Mikami, Mitsuharu Yoshiyama, and Masayuki Takeda Shuqi DuShuqi Du More articles by this author , Isao ArakiIsao Araki More articles by this author , Yuki MikamiYuki Mikami More articles by this author , Mitsuharu YoshiyamaMitsuharu Yoshiyama More articles by this author , and Masayuki TakedaMasayuki Takeda More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)31222-9AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "994: Amiloride-Sensitive Ion Channels in Urinary Bladder Epithelium Involved in Mechanosensory Transduction by Modulating Stretch-Evoked ATP Release." The Journal of Urology, 177(4S), pp. 328–329 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 328-329 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Shuqi Du More articles by this author Isao Araki More articles by this author Yuki Mikami More articles by this author Mitsuharu Yoshiyama More articles by this author Masayuki Takeda More articles by this author Expand All Advertisement PDF downloadLoading ...

Amiloride-Sensitive Ion Channels in Urinary Bladder Epithelium Involved in Mechanosensory Transduction by Modulating Stretch-Evoked Adenosine Triphosphate Release

To examine the possibility that mechanosensitive ion channels, including epithelial Na+ channels (ENaC), are implicated in mechanosensory transduction of the rat urinary bladder. Cystometry with continuous infusion was performed to investigate the effect of intravesically perfused amiloride (a blocker of ENaC) on micturition reflex in urethane-anesthetized female rats. Bladder strips with or without epithelium suspended in organ bath were subjected to varying degrees of mechanical stretch (up to 50%). A luciferin-luciferase assay was used to quantify the change of stretch-evoked adenosine triphosphate (ATP) release by amiloride. The ENaC gene expression was assessed by reverse transcriptase-polymerase chain reaction. The expression and localization of ENaC proteins was examined using immunofluorescent staining. The intravesical perfusion of 1 mM amiloride significantly reduced the frequency of reflex voiding during bladder filling. This effect of amiloride was reversible by washing out the drug. The peak amplitude of micturition pressure was not affected by amiloride. The stretch-evoked ATP release, most of which (more than 90%) came from epithelial layer, was greatly diminished by 1 mM amiloride (from 443 to a 22% increase from basal level at 50% stretch of the original length). The alpha, beta, and gamma-ENaC subunit proteins and genes were expressed in the rat bladder epithelium. The amiloride-sensitive mechanosensitive channel, including ENaC, expressed in the rat bladder epithelium might be involved in the mechanosensory transduction mechanism by controlling stretch-evoked ATP release.

Patch clamp study of the UNC-105 degenerin and its interaction with the LET-2 collagen in Caenorhabditis elegans muscle.

Degenerins have emerged from genetic studies in Caenorhabditis elegans as candidate mechanically gated amiloride-sensitive ion channels for transducing mechanical stimuli into cellular responses. In C. elegans muscle, the existence of a genetic interaction between the unc-105 degenerin gene and let-2, a gene encoding an alpha2(IV) collagen, raised the possibility that UNC-105 may function as a mechanically gated channel in a stretch receptor complex. However, to date, ion channel activity of UNC-105 has only been recorded in a gain-of-function mutant form in heterologous expression systems. In this study we investigated the in situ properties of UNC-105 using the whole cell configuration of the patch clamp technique on body wall muscle cells from acutely dissected C. elegans. Amiloride was found to be without effect on membrane potential of wild-type muscle cells, suggesting that the UNC-105 degenerin is electrically silent in resting muscle. Hypo-osmotic shocks induced a reversible depolarization of muscle cells but which was not affected by amiloride. Deformation of the cells by applying tension to the filamentous complex on which muscle cells remained attached or by ejecting external solution under pressure failed to induce any change of membrane potential. In gain-of-function unc-105(n506) mutant cells, an amiloride-sensitive inward Na(+) current was found to be constitutively active, leading to maintained muscle depolarization. An associated mutation in the alpha2(IV) collagen LET-2 led to the closure of the mutant UNC-105(n506) channel while a collagenase treatment of these double mutant cells caused it to re-open, giving evidence for a functional interaction between LET-2 collagen and mutant UNC-105 channel.

Characterisation of DRASIC in the mouse inner ear

Within the cochlea, the hair cells detect sound waves and transduce them into receptor potential. The molecular architecture of the highly specialised cochlea is complex and until recently little was known about the molecular interactions which underlie its function. It is now clear that the coordinated expression and interplay of hundreds of genes and the integrity of cochlear cells regulate this function. It was hypothesised that transcripts expressed highly or specifically in the cochlea are likely to have important roles in normal hearing. Microarray analyses of the Soares NMIE library, consisting of 1536 cDNA clones isolated from the mouse inner ear, suggested that the expression of the mechanoreceptor DRASIC was enriched in the cochlea compared to other tissues. This amiloride-sensitive ion channel is a member of the DEG/ENaC superfamily and a potential candidate for the unidentified mechanoelectrical transduction channel of the sensory hair cells of the cochlea. The cochlear-enriched expression of amiloride-sensitive cation channel 3 (ACCN3) was confirmed by quantitative real-time polymerase chain reaction. Using in situ hybridisation and immunofluorescence, DRASIC expression was localised to the cells and neural fibre region of the spiral ganglion. DRASIC protein was also detected in cells of the organ of Corti. DRASIC may be present in cochlear hair cells as the ACCN3 transcript was shown to be expressed in immortalised cell lines that exhibit characteristics of hair cells. The normal mouse ACCN3 cDNA and an alternatively spliced transcript were elucidated by reverse transcription polymerase chain reaction from mouse inner ear RNA. This transcript may represent a new protein isoform with an as yet unknown function. A DRASIC knockout mouse model was tested for a hearing loss phenotype and was found to have normal hearing at 2 months of age but appeared to develop hearing loss early in life. The human homologue of ACCN3, acid-sensing ion channel 3, maps to the same chromosomal region as the autosomal recessive hearing loss locus DFNB13. However, we did not detect mutations in this gene in a family with DFNB13 hearing loss.

Activation of neurons in trigeminal caudalis by noxious oral acidic or salt stimuli is not reduced by amiloride

We investigated the possible role of amiloride-sensitive ion channels of the ENaC/DEGenerin superfamily in the activation of trigeminal nociceptive neurons elicited by noxious chemical stimulation of the oral mucosa using two methodologies, single-unit recording and c-fos immunohistochemistry. In pentobarbital-anesthetized rats, single-unit recordings were made from neurons in superficial laminae of dorsomedial trigeminal subnucleus caudalis (Vc) that responded to noxious thermal and chemical stimuli applied to the dorsal tongue. Successive application of each of three chemicals (250 mM pentanoic acid, n=6 units; 250 mM citric acid, n=8; 5 M NaCl, n=6) evoked responses that were not affected following topical application of amiloride (1 mM). In separate experiments, pentobarbital-anesthetized rats received one of the following stimuli delivered to the dorsal tongue: 250 mM pentanoic acid ( n=6); 1 mM amiloride followed by 250 mM pentanoic ( N=6); 5 M NaCl ( n=5); or 1 mM amiloride followed by 5 M NaCl ( n=5). Two hours later they were perfused with 4% paraformaldehyde and the brain stems processed for c-fos immunoreactivity. Both pentanoic acid and 5 M NaCl evoked similar numbers and patterns of fos-like immunoreactivity (FLI) in dorsomedial Vc and other brain stem regions, with no significant difference in counts of FLI in animals pretreated with amiloride. These results suggest that amiloride-sensitive Na + channels are not essential in mediating the activation of intraoral trigeminal nociceptors.

Putative immunolocalization of the mechanoelectrical transduction channels in mammalian cochlear hair cells.

Hair cells bear an apical bundle of stereocilia arranged in serried rows. Deflection of the bundle controls the opening and closing of mechanoelectrical transduction channels, thereby altering the conductance across the apical plasma membrane. Two locations for these channels have been proposed in the bundle, either near the bases of the stereocilia or towards their tips. One hypothesis that is consistent with the latter possibility suggests that fine extracellular filaments, which run between the tips of the shorter stereocilia and the sides of the taller stereocilia behind, operate the channels. Determining the precise position of the channels is essential to test this hypothesis. We have therefore attempted to localize them immunocytochemically. Because hair-cell transduction is amiloride sensitive, the channels may have an amiloride-binding site associated with them. We have therefore used a polyclonal antibody raised against another amiloride-sensitive ion channel to hunt for them. This antibody recognizes a 62-64 kDa band in immunoblots of cochlear tissue, and produces discrete labelling in the hair bundle. This is most concentrated just below the tips of the shorter stereocilia, coinciding with a region of specialization in the closely apposed membranes of the short and tall stereocilia but not with either end of the tip link.

Epithelial Na Channels: The Nature of the Conducting Pore

Na-selective, amiloride-sensitive ion channels are a crucial component of the system for controlled NaCl reabsorption across tight epithelia. A kinetic model for the process of Na conduction through the channel is presented. The model is based on a view of the possible geometry of the pore obtained from an analysis of voltage-dependent block of Na currents by impermeant cations. It is also consistent with the known electrophysiological properties of the channel, including single-channel currents, current-voltage relationships and saturation. The model, while not a unique explanation of the available data, may serve as a starting point for more detailed experimental and theoretical studies.