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A variety of studies have shown that Na(+) reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which alpha(1)-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na(+) channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 microM) decreased ENaC currents across Xenopus laevis oocytes injected with human alpha,beta,gamma-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO(-)), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO(-)-treated A1AT (1 microM) in C57BL/6 mice also decreased Na(+)-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO(-)-treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na(+)-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC), the rate-limiting protein in transepithelial Na(+) vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human alpha-, beta-, and gamma-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive Na(+) currents and gamma-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either alpha- or gamma-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKCalpha/beta1 and PKCzeta. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKCalpha/beta1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells.

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of delta-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the delta-subunit may form multimeric channels with alpha beta gamma-ENaC. Reverse transcription-PCR revealed that delta-ENaC is co-expressed with alpha beta gamma-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that delta-ENaC is co-expressed with alpha-, beta-, and gamma-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for delta alpha beta gamma-ENaC differed from those of both alpha beta gamma- and delta beta gamma-ENaC (n = 6). The contribution of the delta subunit to P(Li)/P(Na) ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of delta alpha beta gamma-ENaC differed from those of alpha beta gamma- and delta beta gamma-ENaC. Co-immunoprecipitation of delta-ENaC with alpha- and gamma-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of delta-ENaC with other subunits could account for heterogeneity of native epithelial channels.

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. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward K+ conductance approximately fourfold greater than that of Na+, Li+, and Ca2+. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas.

The delta-subunit of epithelial Na(+) channels (ENaC) is predominately expressed in brain, heart, and pancreas. The amiloride sensitivity, Na(+) conductance, and critical domains for gating are characterized as a cross between proton-activated Na(+) channels and alpha-ENaC. The hypothesis that external protons may activate human delta-ENaC was addressed by expressing deltabetagamma-hENaC in Xenopus oocytes and evaluating proton-activated current with the two-electrode voltage clamp technique. Our results showed that protons transiently evoked a Na(+) current with an EC(50) of pH 6 overlapped on the basal current of deltabetagamma-hENaC. Proton-activated current was not observed in uninjected oocytes. Studies on gating kinetics revealed that activation, desensitization, and recovery times of proton-activated Na(+) current were 3.8 +/- 0.5 s, 253 +/- 9.5 s, and 10 +/- 3.6 s, respectively (n = 4-12). Alkali metal cation selectivity of the proton-activated current was identical to that of the basal current of deltabetagamma-hENaC. The metabolic acids, lactate, pyruvate, and formate, modified the proton-activated current, as did hypo-osmotic stress. EDTA, hypo-osmolarity, and lactate enhanced proton activation synergistically. Our results suggest that delta-hENaC subunit is essential for proton-activated current and gamma-subunit may potentially regulate the response of delta-hENaC to protons. We have concluded that deltabetagamma-hENaC is a proton-activated cation channel whose closing gate can be regulated by a proton-induced conformational change. Proton-sensitivity of deltabetagamma-hENaC may be an important mechanism for integrating external ischemic signals in inflamed and hypoxic tissues.

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. Whole-cell and single channel results showed that the permeation ratio of Li(+) and Na(+) (P(Li)/P(Na)) for the swap alpha chimeras co-expressed with betagamma-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of P(Li)/P(Na) for the swap delta constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the gamma(Na) of alpha-ENaC but decreased the gamma(Na) of delta-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (K(i)(amil)) was observed in the alpha chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of K(i)(amil) was obtained in the chimeric delta constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the P(Li)/P(Na) ratio, single channel Na(+) conductance, and amiloride sensitivity of alpha- and delta-ENaCs.

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in alpha-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal beta- and gamma-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, alpha-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.

Both extracellular and intracellular proteases can activate epithelial Na(+) channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified alpha-, beta-, and gamma-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 microg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the beta- and gamma-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 microg/ml trypsin resulted in a reduction in the size of the beta- and gamma-transcripts by 7-8 kDa. By using N- and C-terminally truncated beta- and gamma-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 microg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.