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

Abstract

Activity of the epithelial Na ؉ channel (ENaC) is ratelimiting for Na ؉ (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, ␣, ␤, and ␥, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2␣:1␤:1␥ stoichiometry and a higher ordered channel with a 3␣:3␤:3␥ stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of ϳ90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3␣:3␤:3␥ stoichiometry.Ion channels are integral membrane proteins that form selective pores in the plasma membrane through which ions move down their electrochemical gradients. The epithelial Na ϩ channel (ENaC) 1 is a member of the ENaC/Deg gene superfamily (1-4). This family contains several ion channels (e.g. ASIC, BNaC, DEG, DRASIC, ENaC, and FaNaCH) of similar structure that serve diverse physiological functions from modulating mechanosensory transduction and neuronal signaling to control of electrolyte movement across epithelial barriers.Functional ENaC, similar to most other ion channels, is a heteromeric protein complex containing several distinct channel subunits. Four ENaC subunits have been identified, ␣, ␤, ␥, and ␦ (1, 5, 6). The first three subunits are widely expressed and together form the channel resident to the luminal plasma membrane of epithelial cells capable of electrogenic Na ϩ (re-)absorption. Consistent with this idea are findings showing that co-expression of only two of these three subunits produces little to no current in heterologous expression systems (1,5,7,8). The ␦ENaC subunit localizes to the brain and reproductive tissues where it is believed to substitute for ␣ENaC in the functional channel (6). Importantly, the stoichiometric relationship between ENaC subunits is uncertain.Each ENaC subunit has a cytosolic NH 2 and COOH terminus with two transmembrane domains separated by a single large extracellular loop with all three ENaC subunits, ␣, ␤, and ␥, contributing the functional pore (1, 5, 9, 10). Several laboratories have tested ENaC subunit stoichiometry with a biophysical approach ultimately using MacKinnon's formulation to est...

Acid-sensing ion channel (ASIC) 1a/2a heteromers have a flexible 2:1/1:2 stoichiometry

“…In agreement, several studies reported that the related epithelial Na + channel (ENaC) is composed of four (18)(19)(20)(21) or nine subunits (22)(23)(24). In strong contrast, however, the crystal structure of chicken ASIC1 revealed a number of three subunits (12,25), suggesting that all ASICs and their relatives like ENaC are trimers.…”

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

“…Another involved analysis of the sizes of intramembranous particles visualized by freeze fracture followed by electron microscopy (11). Notably, a trimer-of-trimers structure would also explain coimmunoprecipitation and FRET data, showing that ENaC contains at least two copies of each subunit (31). Evidence for the functional relevance of a 'triple-barrel' organization of ENaC has also been presented, indicating that the trimer-of-trimers structure reported here might be physiologically relevant (32).…”

Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

“…Our experimental design and rationale for determining subunit stoichiometry closely followed that of Zheng and colleagues [16,17], Staruschenko and colleagues [13], and Kerschensteiner and colleagues [19]. Zheng et al (2002) and Kerschensteiner et al (2005) successfully used a FRET method in the determination of a fixed stoichiometry of 3:1 for the rod cyclic-nucleotide gated channels and heteromeric Kv2.1/Kv9.3 channels, respectively.…”

“…The efficiency of energy transfer falls off with the sixth power of the distance between the donor and acceptor molecules [14]. FRET has been successfully employed as a molecular ruler in investigations of the stoichiometry, assembly, and conformational arrangement of many types of receptors and channels [13,[15][16][17][18][19]. Another advantage of FRET over biochemistry approaches such as co-IP is that FRET measurement is non-invasive, allowing determination of subunit stoichiometry from intact channels in the cell membrane.…”

“…We found that significant FRET signals were yielded between the three different ASIC subunits, which further indicated that heteromeric ASIC 1a/2a, ASIC 2a/ 2b and ASIC 1a/2b could form. [11,13]. Here we used a FRET method to test the stoichiometry of ASIC channels.…”