Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.

The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure.

Physical and functional interactions between a glioma cation channel and integrin-β1 require α-actinin.

Major plasma membrane components of the tumor cell, ion channels, and integrins play crucial roles in metastasis. Glioma cells express an amiloride-sensitive nonselective cation channel composed of acid-sensing ion channel (ASIC)-1 and epithelial Na(+) channel (ENaC) α- and γ-subunits. Inhibition of this channel is associated with reduced cell migration and proliferation.

ENaCs and ASICs as therapeutic targets.

The epithelial Na(+) channel (ENaC) and acid-sensitive ion channel (ASIC) branches of the ENaC/degenerin superfamily of cation channels have drawn increasing attention as potential therapeutic targets in a variety of diseases and conditions. Originally thought to be solely expressed in fluid absorptive epithelia and in neurons, it has become apparent that members of this family exhibit nearly ubiquitous expression. Therapeutic opportunities range from hypertension, due to the role of ENaC in maintaining whole body salt and water homeostasis, to anxiety disorders and pain associated with ASIC activity.

Glioma-specific cation conductance regulates migration and cell cycle progression.

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation.

Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes.

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits.

Proteolytic cleavage of human acid-sensing ion channel 1 by the serine protease matriptase.

Acid-sensing ion channel 1 (ASIC1) is a H(+)-gated channel of the amiloride-sensitive epithelial Na(+) channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases.

Amiloride docking to acid-sensing ion channel-1.

Amiloride is a small molecule diuretic, which has been used to dissect sodium transport pathways in many different systems. This drug is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as sodium/hydrogen antiporters and sodium/calcium exchangers. The exact structural basis for these interactions has not been elucidated as crystal structures of these proteins have been challenging to obtain, though some involved residues and domains have been mapped.

Knockdown of ASIC1 and epithelial sodium channel subunits inhibits glioblastoma whole cell current and cell migration.

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, alphaENaC, and gammaENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits.

Psalmotoxin-1 docking to human acid-sensing ion channel-1.

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated.

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.

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.

Amiloride-sensitive Na+ channels contribute to regulatory volume increases in human glioma cells.

Despite intensive research, brain tumors remain among the most difficult type of malignancies to treat, due largely to their diffusely invasive nature and the associated difficulty of adequate surgical resection. To migrate through the brain parenchyma and to proliferate, glioma cells must be capable of significant changes in shape and volume. We have previously reported that glioma cells express an amiloride- and psalmotoxin-sensitive cation conductance that is not found in normal human astrocytes.

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.

Surface expression of ASIC2 inhibits the amiloride-sensitive current and migration of glioma cells.

Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current does not exist in normal astrocytes or low grade tumor cells.

Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes.

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca(2+) ([Ca(2+)](i)) and intracellular pH (pH(i)) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a major regulator of [Ca(2+)](i).

Cation selectivity and inhibition of malignant glioma Na+ channels by Psalmotoxin 1.

Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits Na+ currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an IC50 of 36 +/- 2 pM.

ENaC subunit-subunit interactions and inhibition by syntaxin 1A.

Amiloride-sensitive epithelial Na(+) channels (ENaCs) are subject to modulation by many factors. Recent data have also linked the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery to this regulation of ENaC, but the molecular mechanisms that underlie this modulation are poorly understood. In this study, we demonstrate that syntaxin 1A physically interacts with ENaC and functionally regulates ENaC activity.

Mutations in the extracellular loop of alpha-rENaC alter sensitivity to amiloride and reactive species.

We studied the effects of two mutations of the extracellular loop of the alpha-subunit of the (ENaC) on amiloride-sensitive current in Xenopus laevis oocytes and the inhibition of this current by 3-morpholinosydnonimine (SIN-1). Injection of oocytes with wild-type (wt) alpha-,beta-,gamma-rENaC cRNA (8.3 ng/subunit) resulted 48-72 h later in inward Na(+) currents (-5.

The role of Pre-H2 domains of alpha- and delta-epithelial Na+ channels in ion permeation, conductance, and amiloride sensitivity.

Epithelial Na(+) channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na(+) selectivity, unitary Na(+) conductance (gamma(Na)), and amiloride sensitivity of delta-ENaC are strikingly different from those of alpha-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of delta- and alpha-ENaCs.

Molecular cloning and characterization of human acid sensing ion channel (ASIC)2 gene promoter.

Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na(+)-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (-1551 to +117) was cloned and characterized.

Acid-sensing ion channels in malignant gliomas.

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells.

Relationship between a HCO3- -permeable conductance and a CLCA protein from rat pancreatic zymogen granules.

Ca(2+)-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca(2+)-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca(2+)-activated anion conductance in rat pancreatic ZG membranes (ZGM).