Degenerin/epithelial Na + Channel Research Articles

The mechanoreceptor DEG‐1 regulates cold tolerance in Caenorhabditis elegans

Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH‐1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor‐expressing ASG. Ca2+ imaging revealed that ASG neurons respond to warm temperature via the mechanoreceptor DEG‐1, a degenerin/epithelial Na+ channel (DEG/ENaC), which in turn affects downstream AIN and AVJ circuits. Ectopic expression of DEG‐1 in the ASE gustatory neuron results in the acquisition of warm sensitivity, while electrophysiological analysis revealed that DEG‐1 and human MDEG1 were involved in warm sensation. Taken together, these results suggest that cold tolerance is regulated by mechanoreceptor‐mediated circuit calculation.

Functional features of the "finger" domain of the DEG/ENaC channels MEC-4 and UNC-8.

UNC-8 and MEC-4 are two members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of voltage-independent Na+ channels that share a high degree of sequence homology and functional similarity. For example, both can be hyperactivated by genetic mutations [UNC-8(d) and MEC-4(d)] that induce neuronal death by necrosis. Both depend in vivo on chaperone protein MEC-6 for function, as demonstrated by the finding that neuronal death induced by hyperactive UNC-8 and MEC-4 channels is prevented by null mutations in mec-6. UNC-8 and MEC-4 differ functionally in three major ways: 1) MEC-4 is calcium permeable, whereas UNC-8 is not; 2) UNC-8, but not MEC-4, is blocked by extracellular calcium and magnesium in the micromolar range; and 3) MEC-6 increases the number of MEC-4 channels at the cell surface in oocytes but does not have this effect on UNC-8. We previously reported that Ca2+permeability of MEC-4 is conferred by the second transmembrane domain. We show here that the extracellular "finger" domain of UNC-8 is sufficient to mediate inhibition by divalent cations and that regulation by MEC-6 also depends on this region. Thus, our work confirms that the finger domain houses residues involved in gating of this channel class and shows for the first time that the finger domain also mediates regulation by chaperone protein MEC-6. Given that the finger domain is the most divergent region across the DEG/ENaC family, we speculate that it influences channel trafficking and function in a unique manner depending on the channel subunit.

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.

Towards the Identification of the Neuropeptide Binding Site of Hydra Na+ Channels (HyNaCs)

The DEG/ENaC (Degenerin/Epithelial Na+ Channel) ion channel family is comprised of channels that are activated by diverse stimuli such as mechanical stress, peptides or other ligands. The best known ligand-activated family members are the Acid-Sensing Ion Channels (ASICs), which are activated by protons. Interestingly, the closely related Hydra Na+ channels (HyNaCs) from the freshwater polyp Hydra magnipapillata are activated by much more complex ligands, namely the neuropeptides Hydra RFamides I and II. The bona fide ligand binding sites of both ASICs and HyNaCs are unknown. In this study, we aimed to identify the neuropeptide binding site on heteromeric HyNaC2/3/5 to explore how ligands activate this DEG/ENaC ion channel. To identify the binding site, we combined molecular modelling with site-directed mutagenesis. Mutant ion channels were subsequently expressed in Xenopus laevis oocytes and Hydra RFamide I affinity was determined by two-electrode voltage clamp. Molecular modelling suggested a Hydra RFamide I binding site in a region on subunit 3 of a HyNaC2/3/5 homology model that corresponds to the acidic pocket described in the crystal structure of ASIC1. When four residues of HyNaC3 surrounding the suggested binding site were mutated individually, the affinity of Hydra RFamide I was strongly (3.5 to >100-fold for each mutant) reduced. In contrast, less conservative mutations of the corresponding residues in HyNaC2 or HyNaC5 left the peptide affinity relatively unaffected (<3.5-fold reduced for all mutants). In summary, our results suggest that Hydra RFamide I binds to a single binding site on subunit 3 of HyNaC2/3/5 in a region homologous to the acidic pocket of ASIC1, highlighting the importance of this region for ligand binding in DEG/ENaC ion channels.

Computational elucidation, mutational and hot spot-based designing of potential inhibitors against human acid-sensing ion channels (hASIC-1a) to treat various physiological conditions

Acid-sensing ion channels are ligand/proton-gated ion channels belonging to the family of the degenerin/epithelial Na+ channel (DEG/ENaC). They function as a sodium-selective pore for Ca2+ entry into neuronal cells during pathological conditions. The blocking of this channel has therapeutic importance, because at basal physiological pH (7.2), it is in a closed state and under a more acidic condition, and the ASIC1a ion channel is activated. To investigate the different states of the hASIC1a channel based on mutational analysis, structure-based virtual screening and molecular dynamics simulation studies. The system showed stability after 30 ns (after 1500 frame), and it was stabilized to an average value around 2.2Å. During the simulation, the ion channel residues in persistent contact with toxin PcTx1 were D237, E238, D347, D351, E219 and E355. These residues are important physiologically for the activation of the channel. From in silico alanine scanning, the significant hotspots obtained in hASIC1 are E344, P347, F352, D351, E355 and E219. From the sitemap analysis, it was evident that the sitemap found one of the active sites at the PcTx1 binding site with a site score of 1.086 and a D-score of 1.035 for hASIC1. We obtained a few promising hits and final potential hits from the virtual screening in hASIC1 that made interactions with the residues in the acidic pocket (E344, P347, F352, D351, E355 and E219). Based on these studies, the hits and scaffolds of potential therapeutic interest against various pathological conditions are associated with hASIC1a for future studies.

Afferent arteriolar responses to β,γ-methylene ATP and 20-HETE are not blocked by ENaC inhibition

Afferent arteriolar myogenic and tubuloglomerular feedback responses are critical for the proper maintenance of renal hemodynamics and water and electrolyte homeostasis. Adenosine triphosphate (ATP) P2X receptor activation and 20-hydroxyeicosatetraenoic acids (20-HETE) have been implicated in afferent arteriolar autoregulatory responses. Besides these two participants, members of the degenerin/epithelial Na+ channel (DEG/ENaC) family have been demonstrated to play a pivotal role in the afferent arteriolar myogenic response. The aim of this study was to determine if ENaC contributes to P2X receptor- or 20-HETE-mediated afferent arteriolar vasoconstriction. As previously demonstrated, afferent arteriolar diameter responses to increasing perfusion pressure from 100 to 160 mmHg were abolished by ENaC inhibitors amiloride or benzamil. Afferent arteriolar diameter decreased by 29% under control conditions and by 1% and 5% in the presence of amiloride or benzamil, respectively. The P2X receptor agonist β,γ-methylene ATP decreased afferent arteriolar diameter by 3 ± 1%, 7 ± 1%, 12 ± 2%, and 17 ± 3% in response to 0.1, 1, 10, and 100 μmol/L, respectively. ENaC inhibition did not alter the afferent arteriolar vasoconstrictor response to the P2X receptor agonist β,γ-methylene ATP. Like P2X receptor activation, 20-HETE dose-dependently decreased afferent arteriolar diameter and this vasoconstrictor response was not altered by the presence of ENaC inhibitors amiloride or benzamil. These results suggest that DEG/ENaC channels are required for afferent arteriolar autoregulatory responses; however, DEG/ENaC channels do not contribute to P2X receptor- or 20-HETE-mediated afferent arteriolar vasoconstriction.

Gravity Force Transduced by the MEC-4/MEC-10 DEG/ENaC Channel Modulates DAF-16/FoxO Activity in Caenorhabditis elegans

The gravity response is an array of behavioral and physiological plasticity elicited by changes in ambient mechanical force and is an evolutionarily ancient adaptive mechanism. We show in Caenorhabditis elegans that the force of hypergravity is translated into biological signaling via a genetic pathway involving three factors: the degenerin/epithelial Na(+) channel (DEG/ENaC) class of mechanosensory channels of touch receptor neurons, the neurotransmitter serotonin, and the FoxO transcription factor DAF-16 known to regulate development, energy metabolism, stress responses, and aging. After worms were exposed to hypergravity for 3 hr, their muscular and neuronal functions were preserved, but they exhibited DAF-16::GFP nuclear accumulation in cells throughout the body and accumulated excess fat. Mutations in MEC-4/MEC-10 DEG/ENaC or its partners MEC-6, MEC-7, and MEC-9 blocked DAF-16::GFP nuclear accumulation induced by hypergravity but did not affect DAF-16 response to other stresses. We show that exogenous serotonin and the antidepressant fluoxetine can attenuate DAF-16::GFP nuclear accumulation in WT animals exposed to hypergravity. These results reveal a novel physiological role of the mechanosensory channel, showing that the perception of mechanical stress controls FoxO signaling pathways and that inactivation of DEG/ENaC may decouple mechanical loading and physiological responses.

UNC-1 Regulates Gap Junctions Important to Locomotion in C. elegans

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

Chapter 6 - ENaC Proteins in Vascular Smooth Muscle Mechanotransduction

Mechanotransduction influences many aspects of biological function. In the cardiovascular system, mechanotransduction has a significant impact on vascular function. Multiple signal transduction pathways participate in the transmission of biomechanical forces into cellular signals, including integrins, adhesion molecules, cytoskeleton, and activation of membrane-bound transporters and ion channels. This chapter discusses the potential role of a novel family of ion channels with evolutionarily conserved involvement in mechanotransduction, the degenerin/epithelial Na(+) channel (DEG/ENaC) family. Members of the family are identified in a diverse range of species and tissue types. Most members of the DEG/ENaC family form cation selective ion channels, some of which are believed to form mechanically gated ion channels. While most research has focused on degenerins in sensory neuron mechanotransduction, emerging evidence suggests ENaC proteins may also participate in vascular-smooth-muscle mechanotransduction. The chapter describes the potential role and physiological importance of ENaC proteins as mechanosensors in vascular smooth muscle.

Myogenic vasoconstriction in mouse renal interlobar arteries: role of endogenous β and γENaC

Mechanosensitive ion channels are thought to initiate pressure-induced vasoconstriction, however, the molecular identity of these channels is unknown. Recent work from our laboratory suggests that members of the Degenerin/Epithelial Na+ Channel (DEG/ENaC) family may be components of the mechanosensitive ion channel complex in vascular smooth muscle (VSM); however, the specific DEG/ENaC proteins mediating myogenic constriction are unknown. The goal of this study is to determine if specific knockdown of beta or gammaENaC, using dominant-negative (DN) or small-interference RNA (siRNA) molecules, inhibits pressure-induced vasoconstriction in mouse renal interlobar arteries. To address this goal, isolated arteries were transiently transfected with beta or gammaENaC DN-cDNA or siRNA molecules. After 24 h, vessels were either 1) cannulated and pressurized for pressure-diameter response curves or 2) dissociated and immunolabeled to determine VSM cell endogenous ENaC protein expression. We found that transfection of betaENaC DN-cDNA or siRNA suppresses beta-, but not gammaENaC protein expression. Similarly, gammaENaC DN-cDNA or siRNA suppresses gamma-, but not betaENaC protein expression. In addition, transfection of beta- or gammaENaC DN-cDNA or siRNA molecules inhibits pressure-induced vasoconstriction, but does not block agonist-induced vasoconstriction. Our results provide the first direct evidence that beta and gammaENaC proteins are essential in mediating myogenic vasoconstriction in mouse renal interlobar arteries.

ENaC proteins are required for NGF-induced neurite growth

Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial Na(+) channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of beta- and gamma-subunits of ENaC, but not alpha-ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced beta- and gamma-ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation.

Domain near TM1 influences agonist and antagonist responses of peptide-gated Na+ channels

A molecular biological approach was used to analyse the importance of different amino acids for ligand activation and for determining the action of amiloride on peptide- (Phe-Met-Arg-Phe-NH(2), FMRFamide)-gated Na(+) channels, members of the degenerin/epithelial Na channel (DEG/ENaC) family. Amiloride is a low-affinity blocker of most DEG/ENa channels, but has an unusual enhancing effect on responses of some of them. Chimeras were expressed in Xenopus oocytes and studied electrophysiologically. Differences in properties of channels from Helix aspersa and Helisoma trivolvis highlighted a sequence of 50 residues of the extracellular domain, near the first transmembrane segment (TM1), that affected sensitivity to FMRFamide, and whether amiloride blocked or enhanced the response to FMRFamide. Comparisons of chimeras prepared from H. aspersa and the extracellular domains of two other species, Aplysia californica and Lymnaea stagnalis and the preparation of further constructs, showed that amino acids 128-134 in the H. aspersa sequence are important in determining the predominant effect of amiloride and influencing the EC(50) of FMRFamide. The results also showed that amino acids in this region are influenced by amino acids in other regions of the extracellular domain so as to affect not only the magnitude of responses, but also their time course and desensitisation.

Stomatin Modulates Gating of Acid-sensing Ion Channels

Acid-sensing ion channels (ASICs) are H(+)-gated members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family in vertebrate neurons. Several ASICs are expressed in sensory neurons, where they play a role in responses to nociceptive, taste, and mechanical stimuli; others are expressed in central neurons, where they participate in synaptic plasticity and some forms of learning. Stomatin is an integral membrane protein found in lipid/protein-rich microdomains, and it is believed to regulate the function of ion channels and transporters. In Caenorhabditis elegans, stomatin homologs interact with DEG/ENaC channels, which together are necessary for normal mechanosensation in the worm. Therefore, we asked whether stomatin interacts with and modulates the function of ASICs. We found that stomatin co-immunoprecipitated and co-localized with ASIC proteins in heterologous cells. Moreover, stomatin altered the function of ASIC channels. Stomatin potently reduced acid-evoked currents generated by ASIC3 without changing steady state protein levels or the amount of ASIC3 expressed at the cell surface. In contrast, stomatin accelerated the desensitization rate of ASIC2 and heteromeric ASICs, whereas current amplitude was unaffected. These data suggest that stomatin binds to and alters the gating of ASICs. Our findings indicate that modulation of DEG/ENaC channels by stomatin-like proteins is evolutionarily conserved and may have important implications for mammalian nociception and mechanosensation.

The ion channel ASIC1 contributes to visceral but not cutaneous mechanoreceptor function

Visceral mechanoreceptors are critical for perceived sensations and autonomic reflex control of gastrointestinal function. However, the molecular mechanisms underlying visceral mechanosensation remain poorly defined. Degenerin/epithelial Na+ channel (DEG/ENaC) family ion channels are candidate mechanosensory molecules, and we hypothesized that they influence visceral mechanosensation. We examined the influence of the DEG/ENaC channel ASIC1 on gastrointestinal mechanosensory function, on gastric emptying, and on fecal output. We also compared its role in gastrointestinal and somatic sensory function. To assess the role of ASIC1 we studied wild-type and ASIC1-/- mice. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis determined expression of ASIC1 messenger RNA and protein in vagal and spinal sensory ganglia. Colonic, gastroesophageal, and cutaneous afferent fibers were characterized by functional subtype and their mechanical stimulus-response relationships were determined. Gastric emptying was determined by using a 13CO2 breath test. Behavioral tests assessed somatic mechanical and thermal sensitivity. ASIC1 was expressed in sensory ganglia and was lost after disruption of the ASIC1 gene. Loss of ASIC1 increased mechanosensitivity in all colonic and gastroesophageal mechanoreceptor subtypes. In addition, ASIC1-/- mice showed almost double the gastric emptying time of wild-type mice. In contrast, loss of ASIC1 did not affect function in any of the 5 types of cutaneous mechanoreceptors, nor did it affect paw withdrawal responses or fecal output. ASIC1 influences visceral but not cutaneous mechanoreceptor function, suggesting that different mechanisms underlie mechanosensory function in gut and skin. The role of ASIC1 is highlighted by prolonging gastric emptying of a meal in ASIC1-/- animals.

Degenerin/epithelial Na+ channel proteins: components of a vascular mechanosensor.

Mechanosensitive ion channels are thought to mediate stretch-induced contraction in vascular smooth muscle cells (VSMCs); however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although recent reports suggest degenerin/epithelial Na+ channel (DEG/ENaC) proteins may be mechanosensors in sensory neurons, their role as mechanosensors in vascular tissue has not been examined. We first tested whether DEG/ENaC subunits are expressed in cerebral blood vessels and VSMCs and then examined their role as mechanosensors in mediating the myogenic response in intact blood vessels. Using RT-PCR, we found ENaC transcripts expressed in rat cerebral arteries and freshly dissociated rat cerebral VSMCs. We also detected ENaC expression in isolated blood vessels and VSMCs by immunoblotting and immunolocalization. Moreover, inhibition of ENaC with amiloride (1 micromol/L) and benzamil (30 nmol/L, 1 micromol), an amiloride analog, blocked myogenic constriction in isolated rat cerebral arteries. These data suggest that DEG/ENaC proteins are required for vessel responses to pressure and are consistent with the evolutionary conservation of mechanosensory function of DEG/ENaC proteins.

Stomatin immunoreactivity in ciliated cells of the human airway epithelium.

Stomatin is a widely distributed 32kD membrane protein of unknown function. In biochemical studies it is associated with cholesterol+sphingomyelin-rich 'rafts' in the cytomembrane. Genetic studies in C. elegans, supported by microscopic studies in mammalian tissue and co-expression studies in oocytes, suggest a functional link with the DEG/ENaC (degenerin/epithelial Na+ channel) superfamily of monovalent ion channels. Since ENaC channels play a prominent role in the physiology of the respiratory epithelium, we have studied the immunolocalization of stomatin in mature and developing human airway epithelium by means of Western blot analysis, immunocytochemistry, and immunoelectron microscopy. Stomatin immunoreactivity (stomatin-IR) was found in the ciliated cells of the conductive airway epithelium in a distinct distribution pattern with the strongest signal along the cilia. Immunogold labelling revealed immunogold particles at the basal bodies, along the cilia, and at the membrane of the microvilli. The presence of stomatin-IR paralleled the stages of ciliogenesis in airway development, and its appearance preceded the elongation of the axoneme and the cilial outgrowth. Due to its presence in the different cellular locations in the ciliated cell, we suggest that stomatin is involved in various cellular functions. From its ultrastructural position, stomatin could be a candidate for a membrane-associated mechanotransducer with a role in the control of ciliary motility. Stomatin as a raft protein might be a microtubule associated protein moving along the outer surface of the microtubules to its terminal site of action in the cilia. Stomatin-IR in microvilli supports the hypothesis of a co-localization with beta- and gamma- ENaC and, in conclusion, their potential functional interaction to control the composition of periciliary mucus electrolytes.

The mechanosensory protein MEC-6 is a subunit of the C. elegans touch-cell degenerin channel.

Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6 (refs 3, 4), that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. Here we show that mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. We propose that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells.

Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel).

Neuronal members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family of cation channels include the mammalian brain Na(+) channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related alphaENaC or betaENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.

Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)

Neuronal members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of cation channels include the mammalian brain Na+ channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related αENaC or βENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.

Epithelial Na+ channels and stomatin are expressed in rat trigeminal mechanosensory neurons.

Caenorhabditis elegans MEC-4 and MEC-10 are subunits of the degenerin/epithelial Na+ channel (DEG/ENaC) ion channel superfamily thought to be associated with MEC-2 (a stomatin-like protein) in a mechanotransducing molecular complex in specialized touch sensory neurons. A key question is whether analogous molecular complexes in higher organisms transduce mechanical signals. To address this question, we selected mechanoreceptors of the rat vibrissal follicle-sinus complex in the mystacial pad and the trigeminal ganglia for an immunocytochemical and molecular biological study. RT-PCR of poly(A+) mRNA of rat trigeminal ganglia indicated that alpha-, beta-, and gamma-ENaC and stomatin mRNA are expressed in rat trigeminal ganglia. Using immunocytochemistry, we found that alpha-, beta-, and gamma-ENaC subunits and stomatin are localized in the perikarya of the trigeminal neurons and in a minor fraction of their termination site in the vibrissal follicle-sinus complex, where longitudinal lanceolate endings are immunopositive. We conclude that alpha-, beta-, and gamma-ENaC subunits as well as the candidate interacting protein stomatin are coexpressed in a mammalian mechanoreceptor, a location consistent with a possible role in mechanotransduction.