Amiloride-sensitive Cation Channel Research Articles

Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc.

While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. Here, we show that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, we show that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh‐dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary.

Caenorhabditis elegans body wall muscles sense mechanical signals with an amiloride-sensitive cation channel

C. elegans uses specialized mechanoreceptor neurons to sense various mechanical cues. However, whether other tissues and organs in C. elegans are able to perceive mechanical forces is not clear. In this study, with a whole-cell patch-clamp recording, we show that body wall muscles (BWMs) in C. elegans convert mechanical energy into ionic currents in a cell-autonomous manner. Mechano-gated ion channels in BWMs are blocked in amiloride or cation-free solutions. A further characterization of physiological properties of mechano-gate ion channels in BMWs and a genetic screening show that mechanosensation in BMWs is not dependent on UNC-105 and well-defined mechano-gated ion channels MEC-4 and TRP-4 in C. elegans. Taken together, our results demonstrate that BWMs in C. elegans function as mechanoreceptors to sense mechanical stimuli with an amiloride-sensitive, non-selective cation channel.

Amiloride-sensitive cation channel 2 genotype affects the response to a carbon dioxide panic challenge.

Until recently, genetic research into panic disorder (PD) has had only limited success. Inspired by rodent research, demonstrating that the acid-sensing ion channel 1a (ASIC1a) is critically involved in the behavioral fear response to carbon dioxide (CO2) exposure, variants in the human homologue gene amiloride-sensitive cation channel 2 (ACCN2) were shown to be associated with PD. However, the relationship between changes in brain pH and ACCN2, as done in rodents by CO2 exposure, has not been investigated yet in humans. Here, we examined this link between the ACCN2 gene and the response to CO2 exposure in two studies: in healthy volunteers as well as PD patients and using both behavioral and physiological outcome measures. More specifically, 107 healthy volunteers and 183 PD patients underwent a 35% CO2 inhalation. Negative affect was assessed using visual analogue scales and the panic symptom list (PSL), and, in healthy volunteers, cardiovascular measurements. The single nucleotide polymorphism rs10875995 was significantly associated with a higher emotional response in PD patients and with an increase in systolic as well as diastolic blood pressure in healthy subjects. In all measurements, subjects homozygous for the T-allele showed a heightened reactivity to CO2. Furthermore, a trend towards an rs685012 genotype effect on the emotional response was found in PD patients. We provide the first evidence that genetic variants in the ACCN2 are associated with differential sensitivity to CO2 in PD patients as well as healthy volunteers, further supporting ACCN2 as a promising candidate for future research to improve current treatment options.

Functional neuroanatomy in panic disorder: Status quo of the research

AIMTo provide an overview of the current research in the functional neuroanatomy of panic disorder.METHODSPanic disorder (PD) is a frequent psychiatric disease. Gorman et al (1989; 2000) proposed a comprehensive neuroanatomical model of PD, which suggested that fear- and anxiety-related responses are mediated by a so-called “fear network” which is centered in the amygdala and includes the hippocampus, thalamus, hypothalamus, periaqueductal gray region, locus coeruleus and other brainstem sites. We performed a systematic search by the electronic database PubMed. Thereby, the main focus was laid on recent neurofunctional, neurostructural, and neurochemical studies (from the period between January 2012 and April 2016). Within this frame, special attention was given to the emerging field of imaging genetics.RESULTSWe noted that many neuroimaging studies have reinforced the role of the “fear network” regions in the pathophysiology of panic disorder. However, recent functional studies suggest abnormal activation mainly in an extended fear network comprising brainstem, anterior and midcingulate cortex (ACC and MCC), insula, and lateral as well as medial parts of the prefrontal cortex. Interestingly, differences in the amygdala activation were not as consistently reported as one would predict from the hypothesis of Gorman et al (2000). Indeed, amygdala hyperactivation seems to strongly depend on stimuli and experimental paradigms, sample heterogeneity and size, as well as on limitations of neuroimaging techniques. Advanced neurochemical studies have substantiated the major role of serotonergic, noradrenergic and glutamatergic neurotransmission in the pathophysiology of PD. However, alterations of GABAergic function in PD are still a matter of debate and also their specificity remains questionable. A promising new research approach is “imaging genetics”. Imaging genetic studies are designed to evaluate the impact of genetic variations (polymorphisms) on cerebral function in regions critical for PD. Most recently, imaging genetic studies have not only confirmed the importance of serotonergic and noradrenergic transmission in the etiology of PD but also indicated the significance of neuropeptide S receptor, CRH receptor, human TransMEMbrane protein (TMEM123D), and amiloride-sensitive cation channel 2 (ACCN2) genes.CONCLUSIONIn light of these findings it is conceivable that in the near future this research will lead to the development of clinically useful tools like predictive biomarkers or novel treatment options.

Meta-Analysis of Differential Connectivity in Gene Co-Expression Networks in Multiple Sclerosis.

Differential gene expression analyses to investigate multiple sclerosis (MS) molecular pathogenesis cannot detect genes harboring genetic and/or epigenetic modifications that change the gene functions without affecting their expression. Differential co-expression network approaches may capture changes in functional interactions resulting from these alterations. We re-analyzed 595 mRNA arrays from publicly available datasets by studying changes in gene co-expression networks in MS and in response to interferon (IFN)-β treatment. Interestingly, MS networks show a reduced connectivity relative to the healthy condition, and the treatment activates the transcription of genes and increases their connectivity in MS patients. Importantly, the analysis of changes in gene connectivity in MS patients provides new evidence of association for genes already implicated in MS by single-nucleotide polymorphism studies and that do not show differential expression. This is the case of amiloride-sensitive cation channel 1 neuronal (ACCN1) that shows a reduced number of interacting partners in MS networks, and it is known for its role in synaptic transmission and central nervous system (CNS) development. Furthermore, our study confirms a deregulation of the vitamin D system: among the transcription factors that potentially regulate the deregulated genes, we find TCF3 and SP1 that are both involved in vitamin D3-induced p27Kip1 expression. Unveiling differential network properties allows us to gain systems-level insights into disease mechanisms and may suggest putative targets for the treatment.

The RS685012 Polymorphism of ACCN2, the Human Ortholog of Murine Acid-Sensing Ion Channel (ASIC1) Gene, is Highly Represented in Patients with Panic Disorder.

Panic disorder (PD) is a disabling anxiety disorder that is characterized by unexpected, recurrent panic attacks, associated with fear of dying and worrying about possible future attacks or other behavioral changes as a consequence of the attacks. The acid-sensing ion channels (ASICs) are a family of proton-sensing channels expressed throughout the nervous system. Their activity is linked to a variety of behaviors including fear, anxiety, pain, depression, learning, and memory. The human analog of ASIC1a is the amiloride-sensitive cation channel 2 (ACCN2). Adenosine A2A receptors are suggested to play an important role in different brain circuits and pathways involved in anxiety reactions. In this work we aimed to evaluate the distribution of ACCN2 rs685012 and ADORA2A rs2298383 polymorphisms in PD patients compared with healthy subjects. We found no association between ADORA2A polymorphism and PD. Instead, the C mutated allele for ACCN2 rs685012 polymorphism was significantly more frequent in patients than in controls. On the contrary, the TT homozygous wild-type genotype and also the ACCN2 TT/ADORA2A CT diplotype were significantly more represented in controls. These results are suggestive for a role of ACCN2 rs685012 polymorphism in PD development in Caucasian people.

Acid-sensing cation channels

In both the CNS and peripheral nervous system (PNS), energy metabolism, neuronal activity, hypoxia, hypercapnia, and inflammation elicit pH fluctuations that may affect neuronal excitability and function. The acid-sensing ion channels (ASICs) are voltage-independent cation channels that are activated by acidic pH and are expressed in neurons throughout the body. ASICs belong to the degenerin/epithelial sodium (Na+) channel (DEG/ENaC) superfamily, which includes several amiloride-sensitive cation channels. ASICs are primarily permeable to Na+ and elicit cell depolarization, leading to secondary intracellular accumulation of calcium (Ca2+). ASICs in peripheral sensory neurons participate in nociception, mechanosensation, and taste transduction. In the CNS, ASICs are involved in synaptic plasticity, learning, fear conditioning, and maintenance of retinal integrity. Studies using ASIC channel blockers or transgenic mice indicate that ASICs may be involved in several neurologic disorders, including pain, cerebral ischemia, seizures, and neurodegeneration. ASICs may also have a role in anxiety and depression. Therefore, ASICs may represent novel targets for potential treatment of these disorders. The structure, function, modulation, and clinical implications of ASICs have been the subjects of several reviews.1–10

Pharmacology of ASIC channels

Acid‐sensing ion channels (ASICs) form a family of voltage‐independent amiloride‐sensitive cation channels that predominantly conduct Na+ ions. ASICs are activated by extracellular acidification within the physiological range, and they form effective proton sensors in both the central and peripheral nervous system, with increasing evidence of expression in non‐neuronal cells as well. Positive modulators include some metal ions, small molecules, neuropeptides, opioid peptides, and a snake toxin, while inhibitors include nonspecific and/or nonsubtype‐specific small molecules and more specific natural peptides isolated from animal venoms. A combination of genetic and pharmacologic approaches using the weakly selective inhibitor amiloride and more selective peptide toxins has revealed the implication of ASIC channels in an increasing number of physiological and pathophysiological processes, most of them associated with extracellular pH fluctuations, ranging from synaptic plasticity, learning, memory, fear, depression, seizure termination, and neuronal degeneration to nociception and mechanosensation. WIREs Membr Transp Signal 2013, 2:155–171. doi: 10.1002/wmts.88Conflict of interest: ML is Senior Vice President and Chief Scientific Officer of Theralpha.For further resources related to this article, please visit the WIREs website.

Genetic Influences on Response to Mood Stabilizers in Bipolar Disorder

Mood stabilizers form a cornerstone in the long-term treatment of bipolar disorder. The first representative of their family was lithium, still considered a prototype drug for the prevention of manic and depressive recurrences in bipolar disorder. Along with carbamazepine and valproates, lithium belongs to the first generation of mood stabilizers, which appeared in psychiatric treatment in the 1960s. Atypical antipsychotics with mood-stabilizing properties and lamotrigine, which were introduced in the mid-1990s, form the second generation of such drugs. The response of patients with bipolar disorder to mood stabilizers has different levels of magnitude. About one-third of lithium-treated patients are excellent responders, showing total prevention of the episodes, and these patients are clinically characterized by an episodic clinical course, complete remission, a bipolar family history, low psychiatric co-morbidity and a hyperthymic temperament. It has been suggested that responders to carbamazepine or lamotrigine may differ clinically from responders to lithium. The main phenotype of the response to mood stabilizers is a degree of prevention against recurrences of manic and depressive episodes during long-term treatment. The most specific scale in this respect is the so-called Alda scale, where retrospective assessment of lithium response is scored on a 0–10 scale. The vast majority of data on genetic influences on the response to mood stabilizers has been gathered in relation to lithium. The studies on the mechanisms of action of lithium and on the neurobiology of bipolar disorder have led to the identification of a number of candidate genes. The genes studied for their association with lithium response have been those connected with neurotransmitters (serotonin, dopamine and glutamate), second messengers (phosphatidyl inositol [PI], cyclic adenosine-monophosphate [cAMP] and protein kinase C [PKC] pathways), substances involved in neuroprotection (brain-derived neurotrophic factor [BDNF] and glycogen synthase kinase 3-β [GSK-3β]) and a number of other miscellaneous genes. There are no published pharmacogenomic studies of mood stabilizers other than lithium, except for one study of the X-box binding protein 1 (XBP1) gene in relation to the efficacy of valproate. In recent years, a number of genome-wide association studies (GWAS) in bipolar disorders have been performed and some of those have also focused on lithium response. They suggest roles for the glutamatergic receptor AMPA (GRIA2) gene and the amiloride-sensitive cation channel 1 neuronal (ACCN1) gene in long-term lithium response. A promise for better elucidating the genetics of lithium response has been created by the formation of the Consortium on Lithium Genetics (ConLiGen) to establish the largest sample, to date, for the GWAS of lithium response in bipolar disorder. The sample currently comprises more than 1,200 patients, characterized by their response to lithium treatment according to the Alda scale. Preliminary results from this international study suggest a possible involvement of the sodium bicarbonate transporter (SLC4A10) gene in lithium response. It is concluded that the pharmacogenetics of response to mood stabilizers has recently become a growing field of research, especially so far as the pharmacogenetics of the response to lithium is concerned. Clearly, the ConLiGen project is a highly significant step in this research. Although the results of pharmacogenetic studies are of significant scientific value, their possible practical implications are yet to be seen.

A Deg/ENaC cation conductance regulates migration and cell cycle progression in gliomas

Glioma cells express a specific amiloride‐sensitive cation channel composed of ASIC1, α‐ and γENaC. As these tumors are highly invasive, we hypothesized that channel activity was engaging an intracellular signaling pathway regulating migration and proliferation. We used a truncated construct (ASIC1Y67X) and shRNA to knockdown ASIC1 and integrin β1 in D54MG glioma cells, and scratch assays, FACS, patch‐clamp, and immunoblotting, to determine knockdown effects. Cells were synchronized by removal of FBS for 48h prior to the experiments. ASIC1 knockdown reduced scratch area by 39 ± 16% (n≥4 ±SD P<0.001), and significantly increased the percentage of cells in the cell cycle G0/G1 phase (by 19 ± 5% (SD) n≥3 P<0.001) as compared to controls at 24h. Expression of two cell cycle inhibitors, p21Cip1 and p27Kip1 increased by 2.5 fold (n≤5) and 3 fold (n≤6), respectively. Phosphorylation of ERK1/2, a key mediator of proliferation and migration was also reduced (by 47 ± 20% (SD) n≥6 P<0.001) as compared to control at 24h. Knockdown of δENaC (not part of the glioma channel complex), had no effect on any measured parameter. Knockout of integrin β1 inhibited the amiloride‐sensitive current by 89±24% (n≥9; ±SD P<0.001). Our data reveal novel interactions between Deg/ENaC channels, the MAPK pathway and integrins that may form a mechanism by which Na+ current regulates cell proliferation and migration. Supported by NIH Grant # DK037206.

Comparison of two types of δENaC channels cloned from human lungs

δENaC has been detected in human lung epithelial cells. Here we cloned a splicing variant, namely, δ2ENaC in human bronchoalveolar epithelial cells. δ2ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1ENaC. δ2ENaC was expressed in both alveolar type I and II cells as revealed by in situ hybridization. To compare the biophysical and pharmacological features of the splicing variant with the δ1βγ channels, we examined the whole‐cell and single channel properties comparing with those of δ1βγ and αβγ channels. Divergent from δ1βγ channels, the macroscopic currents of δ2βγ channels were greater; the apparent Na+ affinity was stronger; the apparent dissociation constant for amiloride (Kiamil) but not Evans blue was smaller; the EC50 value of capsazepine activation was less; and the gating kinetics by protons were markedly faster. The single channel activity, unitary conductance, and open probability of δ2βγ channels were significantly greater compared to δ1βγ channels. The potential longevity of δ2βγ at cell surface, as shown by the faster exocytosis and slower endocytosis could contribute to the enhanced channel activity. Our data demonstrate that the channels composed of the splicing variant segregated from both δ1βγ and αβγ channels by strikingly diverse biophysical/pharmacological properties and regulation by physiological signal. Our results may account for the heterogeneities of native amiloride‐sensitive cation channels in human lung and other epithelial tissues. This work was supported by NIH grants HL87017, HL031197, ES015676, ES017218, and National Natural Science Foundation of China (30971181).

A genome-wide study of panic disorder suggests the amiloride-sensitive cation channel 1 as a candidate gene

Panic disorder (PD) is a mental disorder with recurrent panic attacks that occur spontaneously and are not associated to any particular object or situation. There is no consensus on what causes PD. However, it is recognized that PD is influenced by environmental factors, as well as genetic factors. Despite a significant hereditary component, genetic studies have only been modestly successful in identifying genes of importance for the development of PD. In this study, we conducted a genome-wide scan using microsatellite markers and PD patients and control individuals from the isolated population of the Faroe Islands. Subsequently, we conducted a fine mapping, which revealed the amiloride-sensitive cation channel 1 (ACCN1) located on chromosome 17q11.2-q12 as a potential candidate gene for PD. The further analyses of the ACCN1 gene using single-nucleotide polymorphisms (SNPs) revealed significant association with PD in an extended Faroese case-control sample. However, analyses of a larger independent Danish case-control sample yielded no substantial significant association. This suggests that the possible risk alleles associated in the isolated population are not those involved in the development of PD in a larger outbred population.

Genomic structure, polymorphism and expression of ACCN1 and ACCN3 genes in the horse

A category of cation gate proteins was shown to be present in sensory neurons and act as receptors of protons present in tissues such as muscles. The Amiloride-sensitive Cation Channel, Neuronal (ACCN) gene family is known to play a role in the transmission of pain through specialized pH sensitive neurons. Muscles from horses submitted to strenuous exercises produce lactic acid, which may induce variable pain through ACCN differential properties. The sequences of the equine cDNAs were determined to be 2.6 kb in length with an open reading frame of 1539 bp for ACCN1 and 2.1 kb in length with an open reading frame of 1602 bp for ACCN3. The ACCN1 gene is 990 kb long and contains 10 exons, and the ACCN3 gene is 4.2 kb long and contains 11 exons. The equine ACCN1 and ACCN3 genes have an ubiquitous expression but ACCN1 is more highly expressed in the spinal cord. We identified one alternative ACCN3 splicing variant present in various equine tissues. These mRNA variants may encode two different protein isoforms 533 and 509 amino acids long. Ten single nucleotide polymorphisms (SNPs) were detected for ACCN1; five in the coding and five in the non-coding region, with no amino acid change, while the three SNPs identified in the coding region of the ACCN3 gene introduce amino acid changes. The equine in silico promoter sequence reveals a structure similar to those of other mammalian species, especially for the ACCN1 gene.

Amiloride-sensitive cation channel 2, neuronal (ACCN2; ASIC1)

Amiloride-sensitive cation channel 2, neuronal (ACCN2; ASIC1)

Quantitative Analysis Of DEG/ENaC Subunits Interaction

Members of the DEG/ENaC protein family are voltage-independent, amiloride-sensitive cation channels. This family includes acid sensing ion channels (ASICs), which are involved in sensory function, modulation of learning and memory in the nervous system. Another branch of the DEG/ENaC family includes epithelial sodium channels (ENaCs) that are key players in sodium transport across epithelia. ‘Classical’ ENaCs are formed by three different subunits named α, β and γ. A forth subunit, δ, has been found in humans and other primates.

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

Conditional gene targeting of the ENaC subunit genesScnn1bandScnn1g

Epithelial sodium channels (ENaC) are members of the degenerin/ENaC superfamily of non-voltage-gated, highly amiloride-sensitive cation channels that are composed of three subunits (alpha-, beta-, and gamma-ENaC). Since complete gene inactivation of the beta- and gamma-ENaC subunit genes (Scnn1b and Scnn1g) leads to early postnatal death, we generated conditional alleles and obtained mice harboring floxed and null alleles for both gene loci. Using quantitative RT-PCR analysis, we showed that the introduction of the loxP sites did not interfere with the mRNA transcript expression level of the Scnn1b and Scnn1g gene locus, respectively. Upon a regular and salt-deficient diet, both beta- and gamma-ENaC floxed mice showed no difference in their mRNA transcript expression levels, plasma electrolytes, and aldosterone concentrations as well as weight changes compared with control animals. These mice can now be utilized to dissect the role of ENaC function in classical and nonclassic target organs/tissues.

Lack of association between the amiloride-sensitive cation channel 2 (ACCN2) gene and anxiety spectrum disorders

Ion channels are involved in a wide range of central nervous system functions and have been implicated in several neuropsychiatric disorders. Rodent studies suggest that the acid-sensing ion channel, ASIC1, may play a role in fear conditioning, a model for human anxiety disorders. In this study, we examined, for the first time, the human analog of ASIC1, the amiloride-sensitive cation channel 2 (ACCN2) gene, for its association with genetic risk across a range of anxiety spectrum phenotypes. Using multivariate structural equation modeling, we selected twin pairs scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, generalized anxiety disorder, panic disorder, agoraphobia, and social phobia, from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. One member from each selected pair for whom DNA was available was entered into a 2-stage, case-control association study for the ACCN2 gene. In the resulting sample of 589 cases and 539 controls, a total of seven single nucleotide polymorphisms that represented the major allelic variation across the ACCN2 locus were screened in stage 1, the positive results of which were tested for replication in stage 2. Although several markers or haplotypic combinations met threshold significance criteria in stage 1, their association was not replicated in stage 2. Post hoc analyses did not reveal significant association to the specific psychiatric phenotypes. Although the ACCN2 gene may play a role in rodent fear conditioning, we could not detect association with genetic risk shared among human anxiety spectrum disorders.

Lipopolysaccharides modify amiloride‐sensitive Na+ transport processes across H441 lung epithelial cells

Bacterial infections modify the ion transport processes that control lung fluid homeostasis. We investigated the effects of bacterial lipopolysaccharides (LPS) on amiloride‐sensitive Na+ transport across H441 cells. Monolayers were cultured at air interface on permeable supports for 7 days, mounted in Ussing chambers and bathed in physiological saline. Mean resistance (Rt) was 296 ± 69 Ωcm2, n = 5. Incubation with 15μgml−1 LPS for 8 hours caused a reduction in amiloride‐sensitive Isc (10 μM amiloride) from 29 ± 4 to 20 ± 3 μ Acm−2, p<0.05 n = 5. There was no change in amiloride‐insensitive Isc or resistance. Pre‐treatment of cells with 20μM PD98059 (MAPK inhibitor) prevented the effect of LPS (29 ± 3.0 μAcm−2, p<0.05 n = 6). LPS also shifted the amiloride concentration effect curve from an EC50 for amiloride of 6.8 × 10−7 M in control cells to 6.4 × 10−6 M in LPS treated cells p<0.05, n = 4. Using cell‐attached patch recordings we identified two constitutively active amiloride‐sensitive cation channels in the apical membrane of H441 cells. A 5pS highly Na+ selective ENaC–like channel and an 18pS non‐selective cation channel (NSC). Treatment with LPS increased the frequency of patches containing the less amiloride‐sensitive NSC by over 50%. We suggest that LPS alters the expression of ENaC/NSC channels in H441 cells resulting in a reduced amiloride‐sensitivity and Na+ conductance. Supported by The Wellcome Trust & BBSRC.

Participation of the Chaperone Hsc70 in the Trafficking and Functional Expression of ASIC2 in Glioma Cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.