James K. Bubien

TRIPLE-BARREL ORGANIZATION OF ENAC, A CLONED EPITHELIAL NA+ CHANNEL

A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the α subunit alone and those formed by α, β, and γ subunits. The protein was derived from two sources: either from in vitro translation reaction followed by Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found that either α-rENaC alone or α- in combination with β- and γ-rENaC, produced highly Na+-selective (PNa/PK = 10), amiloride-sensitive (Kiamil = 170 nM), and mechanosensitive cation channels in planar bilayers. α-rENaC displayed a complicated gating mechanism: there was a nearly constitutively open 13-picosiemens (pS) state and a second 40-pS level that was achieved from the 13-pS level by a 26-pS transition. α-, β-, γ-rENaC showed primarily the 13-pS level. α-rENaC and α,β,γ-rENaC channels studied by patch clamp displayed the same gating pattern, albeit with >2-fold lowered conductance levels, i.e. 6 and 18 pS, respectively. Upon treatment of either channel with the sulfhydryl reducing agent dithiothreitol, both channels fluctuated among three independent 13-pS sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude between all three conductance levels. Different combinations of α-, β-, and γ-rENaC in the reconstitution mixture did not produce channels of intermediate conductance levels. These findings suggest that functional ENaC is composed of three identical conducting elements and that their gating is concerted.

The Carboxyl Terminus of the α-Subunit of the Amiloride-sensitive Epithelial Sodium Channel Binds to F-actin

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between α-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of α-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of α-, β-, and γ-ENaC revealed co-localization of α-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with α-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of α-ENaC. A direct interaction between F-actin and the COOH terminus of α-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

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, αENaC, and γENaC 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. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high PK+/PNa+ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K+ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

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. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our previous results have shown that the acid-sensing ion channels (ASICs), members of the epithelial Na+ channel (ENaC)/degenerin (DEG) family of ion channels are part of this current pathway. We hypothesized that one member of the ENaC/DEG family, ASIC2, is retained intracellularly and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high grade gliomas. In this study we show that the chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, inhibit the constitutively activated inward current and reduce cell growth and migration in glioblastoma multiforme. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane, and once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This in turn compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/DEG subunits and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a phenotype resembling that of normal astrocytes.

Epithelial Na+ Channel (ENaC), Hormones, and Hypertension

This minireview examines both the basic science and clinical observations over the past 20 years to show how and why overstimulation of the amiloride-sensitive epithelial Na+ channel (ENaC) expressed by epithelial principal cells of the renal collecting duct may be responsible for a large portion of hypertension in modern society. This idea is based on the finding that, in Liddle syndrome, a mutation of the β- and/or γ-subunits of ENaC produces an activated ion channel, in turn resulting in severe hypertension that is resistant to most forms of conventional antihypertensive therapy. ENaC can also be stimulated to conduct sodium by two hormones: aldosterone and insulin. These hormones are both often elevated in obese individuals with therapy-resistant hypertension. Thus, overstimulation of ENaC by metabolic abnormalities in obese individuals may be a likely cause of the hypertension that accompanies obesity. The molecular mechanisms underlying both Liddle syndrome and obesity-related hypertension are different (i.e. genetic and hormonal, respectively), but both have the same end result, namely increased ENaC activity.

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.

Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl‐ conductance defect in cystic fibrosis lymphocytes by transfection with wild‐type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non‐CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector‐specific sequences. Both fluorescence and patch‐clamp assays showed that transfectants expressing wild‐type CFTR acquired properties previously associated with Cl‐ conductance (GCl) regulation in non‐CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist‐induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild‐type CFTR corrects the defects in Cl‐ conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR‐mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes.

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddles disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine α-ENaC bound to the plasma membrane of normal and Liddles lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddles lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%;p < 0.001) for both normal and Liddles cells after treatment with 40 μm 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddles cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P open was 4.2 ± 3.9% for patches from non-Liddles cells, and 27.7 ± 5.4% in patches from Liddles lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddles disease induces excessive channel expression.

Malignant human gliomas express an amiloride-sensitive Na+ conductance

Human astrocytoma cells were studied using whole cell patch-clamp recording. An inward, amiloride-sensitive Na+ current was identified in four continuous cell lines originally derived from human glioblastoma cells (CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures of cells obtained from glioblastoma multiforme tumors (up to 4 passages). In addition, cells freshly isolated from a resected medulloblastoma tumor displayed this same characteristic inward current. In contrast, amiloride-sensitive currents were not observed in normal human astrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas. The only amiloride-sensitive Na+channels thus far molecularly identified in brain are the brain Na+ channels (BNaCs). RT-PCR analyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 in these tumors and in normal astrocytes. These results indicate that the functional expression of amiloride-sensitive Na+ currents is a characteristic feature of malignant brain tumor cells and that this pathway may be a potentially useful target for therapeutic intervention.

CFTR may play a role in regulated secretion by lymphocytes: a new hypothesis for the pathophysiology of cystic fibrosis.

Abstract. Human lymphocytes and pancreatic acinar cells have a common function: both cell types secrete specific proteins in response to extracellular signals. Acinar cells secrete digestive enzymes, while lymphocytes secrete antibodies and cytokines. Both cell types utilize similar receptor-mediated activation systems, similar signal transduction pathways (i.e., α adrenergic receptors, and cAMP), and express the cystic fibrosis transmembrane conductance regulator (CFTR). Preliminary tests of the hypothesis that B lymphocytes are capable of regulated secretion were carried out using transformed lymphocytes. λ light chain secretion rates were measured in response to treatment with 8-CPT-cAMP. A rapid transient increase in secretion was observed in non-CF lymphocytes. This effect was absent in CF lymphocytes. A failure of regulated secretion could cause a reduced response to antigen presentation, and an inability to completely clear pathogens such as Pseudomonasaeruginosa. Another piece of circumstantial evidence is that lung-transplanted CF patients remain chronically ill. While immunosuppressive therapy may contribute to the chronic illness, the phenomenon is more acute in CF lung-transplant patients than non-CF lung-transplant recipients receiving the same immunosuppressive therapy. A defect in regulated secretion of antibodies and cytokines in response to antigens may be the source of a long suspected, but as yet unproved CFTR-mediated immunological defect underlying the pulmonary morbidity and mortality in cystic fibrosis (CF).