δ-Subunit Confers Novel Biophysical Features to αβγ-Human Epithelial Sodium Channel (ENaC) via a Physical Interaction

Abstract

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 ␦-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na ؉ conductances in epithelial cells. For example, the ␦-subunit may form multimeric channels with ␣␤␥-ENaC. Reverse transcription-PCR revealed that ␦-ENaC is co-expressed with ␣␤␥-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that ␦-ENaC is co-expressed with ␣-, ␤-, and ␥-ENaC in H441 cells at the protein level. Measurement of current-voltage relationships revealed that the cation selectivity ratios for Na ؉ /Li ؉ /K ؉ /Cs ؉ /Ca 2؉ /Mg 2؉ , the apparent dissociation constant (K i ) for amiloride, and unitary conductances for ␦␣␤␥-ENaC differed from those of both ␣␤␥-and ␦␤␥-ENaC (n ‫؍‬ 6). The contribution of the ␦ subunit to P Li /P Na ratio and unitary Na ؉ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC 50 for proton activation, mean open and closed times, and the self-inhibition time of ␦␣␤␥-ENaC differed from those of ␣␤␥-and ␦␤␥-ENaC. Co-immunoprecipitation of ␦-ENaC with ␣-and ␥-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of ␦-ENaC with other subunits could account for heterogeneity of native epithelial channels.

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

“…The ␦ subunit of ENaC replaces the ␣ subunit in the channel complex expressed in some tissues of several species, including humans (but not rodents). Interestingly, human ␦␤␥ channels are poorly inhibited by external Na ϩ (46). The ␦ subunit has a Pro residue at the equivalent site to ␣Asp-365.…”

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

“…The a and d subunits are expressed in human lung tissues and primary alveolar type II cells (26,32). d ENaC-like channels -cyclic monophosphate-Na (CPT-cGMP).…”

“…To elicit self-inhibition, oocytes were held at 260 mV continuously while the bath solutions were switched quickly from ND1 and ND96 solutions, controlled with a SF-77B Perfusion Fast-Step (Warner Ins., Hamden, CT) fluidic exchange system and software pCLAMP as previously described (32,33). CPT-cGMP (AXXORA, LLC, San Diego, CA) stock solution (50 mM) was prepared in water and stored at 220 8 C. MTSET (Toronto Research Chemicals, Downsview, ON, Canada) was freshly prepared every 10 minutes.…”

α1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo

“…Detailed description of these techniques has been previously reported (22)(23)(24). In brief, defolliculated oocytes, isolated from Xenopus laevis frogs, were injected with cRNAs encoding for wild-type a, b, g-hENaC (8.4 ng each), dissolved in 50 nl of RNase-free water per oocyte, and incubated in half-strength L-15 medium for 24 to 48 hours.…”