Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na + channel

the epithelial Na + channel (ENaC) is rate limiting for Na + (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, α , β and γ , 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 α :1 β :1 γ stoichiometry and a higher ordered channel with a 3 α :3 β :3 γ 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 CHO and COS-7 cells. We find that when all three subunits are co-expressed, ENaC contains at least 2 of each type of subunit. We use here a biophysical approach distinct from that used previously by others to distinguish between the 2:1:1 and 3:3:3 subunit stoichiometry proposed for ENaC. Our approach was based on fluorescence resonance energy transfer between ENaC subunits differentially tagged with distinct fluorophores. The current results demonstrate that when all three subunits are co-expressed, ENaC can contain more than one copy of each type of subunit and thus, our findings are more consistent with a higher ordered channel.

We use here a biophysical approach distinct from that used previously by others to distinguish between the 2:1:1 and 3:3:3 subunit stoichiometry proposed for ENaC. Our approach was based on fluorescence resonance energy transfer between ENaC subunits differentially tagged with distinct fluorophores. The current results demonstrate that when all three subunits are co-expressed, ENaC can contain more than one copy of each type of subunit and thus, our findings are more consistent with a higher ordered channel.

EXPERIMENTAL PROCEDURES
Plasmids -Full-length mouse α, β, γENaC subunit cDNAs were ligated in-frame behind either eCFP or eYFP into pECFP-C1 or pEYFP-C1 (Clontech; Palo Alto, CA) at the XhoI and EcoRI restriction endonuclease sites. Initially, channel subunit cDNAs were amplified with standard PCR from the respective pCMV-Myc-ENaC plasmids described previously by our laboratory (16).
For α, β and γENaC, the upstream and downstream primers were 5'- Technology, Brattleboro, VT) and 525-575 nm (eYFP; HQ 550/50 bandpass; Chroma Technology). Preceding the emission filters in the light path was a NFT 515 nm dichroic mirror that split the eCFP and eYFP signals to distinct photomultiplier tubes. This system has been described previously (19). All fluorescence data were collected with a high-resolution (1.4 NA), 60x oil-immersion DIC lens. The software controlled 510 LSM confocal microscope enabled timed regional photobleaching with photobleaching performed selectively at 514 nm with a mean reduction of 525-575 nm (eYFP) emissions to less than 15 % in the photobleached region.
Images were acquired before and after photobleaching.
Percent FRET efficiency between eCFP (donor) and eYFP (acceptor) was quantified at steady-state in fixed cells with acceptor photobleaching methods (also called donor dequenching; (19) using the following formula: where A 0 and A 1 are eCFP emissions in the photobleached region before and after photobleaching, respectively; and B 0 and B 1 are eCFP emissions in a non-bleached region before and after photobleaching, respectively. With this approach, % FRET efficiency is the percent increase in donor emissions upon dequenching following selective acceptor photobleaching with spurious changes in donor emissions normalized to an internal control.
bovine serum supplemented with standard antibiotics and 10 µM amiloride. Co-immunoprecipitation experiments were performed to determine possible interactions between like ENaC subunits. Immunoprecipitation experiments followed published protocols (16;18;20). In brief, cells were co-transfected with Myc-and HA-tagged, as well as fluorophore-

RESULTS
We used, in the current study, a biophysical approach based on FRET between differentially fluorophore-tagged channel subunits to determine ENaC subunit stoichiometry.
This method has been used successfully previously by Zheng and colleagues to determine stoichiometry of rod cyclic-nucleotide-gated (CNG) channels (21) and by Erickson and colleagues to establish that calmodulin is pre-tethered to voltage-gated Ca 2+ channels (22). Our general experimental design and rationale closely followed that of Zheng and colleagues (21).

DISCUSSION
Several laboratories now have investigated the subunit stoichiometry of ENaC. These studies have lead to the proposal of two possible subunit arrangements. Firsov and colleagues (9), Dijkink and colleagues (14), and Kosari and colleagues (12) propose that ENaC is a tetramer with a subunit stoichiometry of 2α:1β:1γ. This is an attractive arrangement for it parallels the stoichiometry of other cation channels, such as K v and voltage-gated Na + and Ca 2+ channels that have a 4-fold internal symmetry (11;23;24). Moreover, for K + channels, such as K ir , comprised of subunits having only two transmembrane domains, a featured shared with all ENaC subunits, the established stoichiometry is a tetramer (25). In contrast to this arrangement, Snyder and colleagues (13) and Eskanderi and colleagues (15) propose that for a cation channel, ENaC has a unique stoichiometry resulting in a higher ordered structure with a possible 3α:3β:3γ arrangement. Interestingly, many of the same methods and experimental approaches were used to provide support for both the 2:1:1 and 3:3:3 stoichiometry. Thus, it has become critical to test ENaC subunit stoichiometry with several independent assays. Here we tested between the two previously proposed models of ENaC subunit stoichiometry using a biophysical approach, distinct from that used previously, dependent on fluorescence resonance energy transfer between ENaC subunits differentially tagged with fluorophores. We find that ENaC contains at least two of each type of subunit. Biochemical results demonstrating that oligomerized ENaC has a molecular mass greater than 600 kDa with subunits alone having masses ~ 90 kDa, and that each ENaC subunit is capable of interacting with like subunits also support the idea that the fully Another advantage of using FRET is that this method yields binary results with respect to determining protein interaction and thus, it simplifies analysis of whether two ENaC subunits interact. Moreover, FRET directly quantifies protein interactions. These two advantages are in contrast to previous studies 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 subsequently fitted by idealized equations to establish subunit relationships (9;12;13). These earlier electrophysiology studies, moreover, required the assumption that mutant effects are dominant. Such an assumption was not necessary with the current approach. In contrast to these earlier studies, however, the current investigation cannot definitively determine absolute subunit stoichiometry but rather determines relative subunit levels with our results suggesting that ENaC contains at least 2 of each type of subunit.
The current study, similar to the earlier electrophysiological probing of ENaC subunit stoichiometry, also assumes that single monomers do not readily form homomeric channels.
Two lines of evidence from the current study and many findings by others support this contention. Firstly, our patch results (table 1) demonstrate that expression of single subunits and co-expression of any two of the three subunits do not readily form functional channels; whereas, co-expression of all three subunits results in abundant functional channels. These observations are consistent with similar findings by others (5;7;27;28). Secondly, FRET between differentially tagged but similar monomers (e.g CFP-γENaC & YFP-γENaC) was not observed.
Thus, formation of homomeric channels is rare and does not interfere to a significant extent with our results.
Another limitation of the approach used in the current study was that it established subunit stoichiometry for the entire cellular pool of ENaC. This is distinct from the earlier electrophysiology studies that investigate stoichiometry for ENaC in the plasma membrane (9;12;13). However, our biochemical results showing that ENaC reconstituted in CHO and COS-7 cells is defined by a single major species that has a molecular mass greater than 600 kDa provides support for the idea that when all three ENaC subunits are co-expressed they quickly and readily oligomerize into the final channel complex. These data argue that the stoichiometry of the total cellular pool of channels is representative of those in the membrane. In addition, our electrophysiology results indicated that tagged ENaC subunits used for FRET reconstitute functional channels demonstrating that they can oligomerize into channels that are functional at the plasma membrane. Finally, our results similar to that of others (5;9;27;28), support the idea that when all three channel subunits are available, ENaC prefers to oligomerize into a heteromeric complex containing each type of subunit.
An alternative interpretation of our results that cannot be excluded with the current data set is that tetrameric ENaC cluster and our results reflect inter-rather then intra-channel subunit FRET. We believe this not to be the case for we see no consistent channel clustering when assaying ENaC reconstituted in CHO cells at the single channel level (16;17). In addition, this  Arrows denote FRET and X denotes a lack of energy transfer.      Fig. 6