Electrophysiological and Biochemical Evidence That DEG/ENaC Cation Channels Are Composed of Nine Subunits

Abstract

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero-and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na ؉ channel (hENaC), we expressed the three wild-type hENaC subunits (␣, ␤, and ␥) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three ␣, three ␤, and three ␥ subunits. Sucrose gradient sedimentation of ␣hENaC translated in vitro, as well as ␣-, ␤-, and ␥hENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.The DEG/ENaC protein family includes channels with diverse physiologic and pathophysiologic functions. Epithelial Na ϩ channels (ENaC) absorb Na ϩ in kidney, lung, and intestine (1, 2), and mutations in human ENaC (hENaC) cause disease (3-6). Several family members from Caenorhabditis elegans, including MEC-4, MEC-10, and DEG-1, play a role in mechanotransduction, and some gain-of-function mutations cause neurodegeneration (7). In Helix aspersa, the FMRFamide-gated channel (FaNaCh) functions as a neurotransmitter receptor (8). Three family members have recently been identified in the mammalian nervous system, BNC1 (MDEG, BNaC1) (9 -11), BNaC2 (ASIC) (11,12), and DRASIC (13).All members of the DEG/ENaC family appear to function as multimers. ENaC contains three homologous subunits, ␣, ␤, and ␥ (14 -18). Functional studies show that simultaneous expression of all three subunits is required to generate maximal Na ϩ current, although expression of ␣ENaC alone can produce small currents. In addition, biochemical data show that the three human ENaC (hENaC) subunits associate (19). Genetic evidence suggests that MEC-4, MEC-10, and DEG-1 also function as heteromultimers (7,20). However, the subunit stoichiometry is not known for any DEG/ENaC channel.EXPERIMENTAL PROCEDURES cDNAs and mutations were generated as described previously (9,17,18). FaNaCh was amplified by polymerase chain reaction following reverse transcription of RNA from H. aspersa. We tagged the C terminus of ␣hENaC with the sequence DYKDDDDK (␣ Flag ) for immunoprecipitation by anti-Flag M2 monoclonal antibody. This did not alter the function of the ␣ subunit in Xenopus oocytes or epithelia or its ability to associate with other subunits (19).Wild-type or mutant ␣-, ␤-, and ␥hENaC (0.2 ng each) were expressed in Xenopus oocytes by nuclear injection of cDNA (18). When a mixture of wild-type and mutant cDNAs for a subunit was coinjected, the total amount of cDNA for the subunit remained constant. 16 -24 h after injection, whole-cell Na ϩ current was measured by two-electrode voltage clamp at Ϫ60 mV (bathing solution, 116 mM NaCl, 2 mM KCl, 0.4 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4). To ...

Fluorescence Resonance Energy Transfer Analysis of Subunit Stoichiometry of the Epithelial Na+ Channel

“…Biochemical results demonstrating that oligomerized ENaC has a molecular mass Ͼ600 kDa with subunits alone having masses ϳ90 kDa and that each ENaC subunit is capable of interacting with similar subunits also support the idea that the fully complexed channel contains more than four subunits. Our FRET and biochemical findings are most consistent with the sucrose gradient and freeze-fracture results of others suggesting a subunit stoichiometry of 3␣:3␤:3␥ (13,15).…”

“…Results from these types of experiments support both a 2␣:1␤:1␥ and a 3␣:3␤:3␥ stoichiometry. Results from biochemical experiments using sucrose gradient sedimentation and quantitative analysis of cell surface expression of ENaC subunits, moreover, provide support for both the 2:1:1 and 3:3:3 stoichiometry (9,13,14). Results testing functionality of ENaC concatamers supporting a 2:1:1 stoichiometry, similarly, contrast with freeze-fracture electron microscopy results supporting a higher ordered stoichiometry (9, 15).…”

“…Moreover, FRET directly quantifies protein interactions. These two advantages are in contrast to previous studies (9,12,13) of ENaC subunit stoichiometry where changes in wild-type and mutant channel activity in response to blockers and modifying reagents were assessed with electrophysiology, and results were subsequently fitted by idealized equations to establish subunit relationships. These earlier electrophysiology studies, moreover, required the assumption that mutant effects are dominant.…”

“…Several laboratories have tested ENaC subunit stoichiometry with a biophysical approach ultimately using MacKinnon's formulation to establish titration and inhibition curves (9,(11)(12)(13). This approach relies on patch clamp electrophysiology to discriminate between channels containing wild-type subunits from those containing mutant subunits with differential sensitivities to channel blockers and cysteine-modifying methanethiosulfonates.…”

Clinical implication of lung fluid balance in the perinatal period

“…ENaC has been proposed to be a heterotetrameric protein (abag), 36 but octameric or nonameric structures have also been suggested. 37,38 The relative importance of these three subunits may vary between species and tissues. For example, in Xenopus laevis oocytes and human kidney, the a-subunit seems to be the critical subunit, 27 whereas the b-ENaC subunit may be the rate-limiting subunit for lung airway epithelial Na þ transport.…”