Differential Assembly of Native ENaC Complexes Across Mouse Epithelial Tissues

1 The epithelial sodium channel (ENaC) governs sodium and fluid absorption in the lung, 2 kidney, and colon, but the organization of native ENaC complexes has remained 3 difficult to define because of their low abundance and biochemical instability. To enable 4 direct analysis of native assemblies, we generated a knock -in mouse in which the 5 endogenous γ subunit is fused at its C terminus to mVenus, a 3C protease cleavage 6 site, and a 3xFLAG epitope (ENaCγ-VF). The tag preserves physiological ENaC 7 function, as ENaCγ-VF mice display normal electrolyte handling, benzamil affinity, and 8 amiloride-sensitive Na⁺:K⁺ responses indistinguishable from wild -type animals. Using 9 fluorescence-detection size-exclusion chromatography and single -molecule pull-down, 10 we directly monitor intact native ENaC complexes from lung, kidney, and colon and 11 uncover marked tissue-to-tissue differences in channel abundance and apparent 12 complex size. Dual-color analysis with a fluorescent Fab against ENaCα marks fully 13 assembled αβγ channels, while γ-based fluorescence reports the broader population of 14 γ-containing assemblies. In combination, the ENaCγ -VF line provides a biochemical 15 anchor for identifying regulatory and trafficking proteins that co -purify with native ENaC 16 complexes. These data show that ENaC architecture in vivo is heterogeneous, and 17 establish ENaCγ-VF mice as a platform for dissecting how epithelial environments 18 shape ENaC assembly, composition, and regulation. 19 20 21 22 23 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 3

24 Communication across epithelial barriers depends critically on the controlled movement 25 of sodium ions, a process in which the epithelial sodium channel (ENaC) plays a central 26 and indispensable role. Situated at the apical membrane of epithelial cells, ENaC drives 27 sodium entry that underlies extracellular fluid balance, blood pressure regulation, and 28 airway surface hydration1-4. Its activity is required for efficient alveolar fluid clearance in 29 the lung5, 6, sodium reabsorption in the distal nephron1, and electrolyte absorption in the 30 colon7. Dysregulation of ENaC function contributes to diseases such as cystic fibrosis8 31 and pseudohypoaldosteronism9-12, as well as multifactorial disorders including 32 pulmonary edema13 and hypertension14-25. Hypertension alone affects more than one 33 billion individuals worldwide and remains the leading modifiable risk factor for 34 cardiovascular disease26-28, chronic kidney disease29, and cognitive decline30. These 35 clinical associations have long underscored the need to understand how ENaC is 36 assembled, regulated, and stabilized within its native environment. 37 38 Before recombinant expression was feasible, early conceptual models of ENaC biology 39 emerged largely from electrophysiological studies of transepithelial transport, which 40 inferred the presence of a selective sodium channel from its amiloride sensitivity and 41 ionic selectivity31. These foundational observations established the physiological role of 42 ENaC long before its molecular identity was known. The subsequent cloning of the α, β, 43 and γ subunits provided the first molecular description for the channel and revealed a 44 family of homologous proteins with conserved topology across vertebrate epithelia32-34. 45 Recombinant expression systems soon enabled functional dissection through 46 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 4 mutagenesis, proteolytic activation studies, and single-channel recordings, offering 47 high-resolution insights into ENaC architecture35-37, gating38, and inhibition39. 48 49 Yet these advances captured only a portion of ENaC biology. Within native tissues, 50 ENaC expression is highly compartmentalized40, its maturation requires sequential 51 cleavage by distinct proteases39, 41, and its surface abundance is dynamically regulated 52 by a network of accessory proteins and ubiquitin ligases1, 42-44. Electrophysiological and 53 transcriptomic analyses have yielded valuable perspectives, but neither directly resolves 54 how ENaC subunits assemble into functional complexes, nor how regulatory factors 55 shape the architecture and stability of those assemblies. As a result, the molecular 56 organization of ENaC in situ, its subunit stoichiometry, tissue-specific variability, and 57 regulatory context, remain only partially defined. A fundamental obstacle has been the 58 absence of tools enabling direct detection, purification, and visualization of ENaC under 59 non-denaturing conditions. ENaC is expressed at exceptionally low abundance, 60 biochemically labile, and embedded within lipid environments that complicate 61 solubilization45-47. Attempts to recover native ENaC have traditionally yielded insufficient 62 quantities for systematic analysis, limiting the field to indirect methods or recombinant 63 models that cannot fully replicate the physiological assembly landscape. 64 65 To address this longstanding bottleneck, we developed a genetically engineered knock-66 in mouse line (ENaCγ-VF) in which the endogenous γ subunit is fused at its C terminus 67 to mVenus, followed by a 3C protease site and a 3xFLAG epitope. This multifunctional 68 tag enables fluorescence-based detection, affinity purification, and gentle protease-69 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 5 mediated elution of intact ENaC complexes while preserving native expression levels, 70 regulatory control, and tissue specificity. Here, we validate that the tagged γ subunit 71 supports normal ENaC function in vivo and demonstrate that the ENaCγ-VF line is 72 compatible with fluorescence-detection size-exclusion chromatography (FSEC)48 and 73 single-molecule pull-down (SiMPull)49, permitting direct quantification of native ENaC 74 assemblies isolated from lung, kidney, and colon. In parallel, we developed a 75 fluorescent Fab fragment targeting ENaCα, enabling dual-color, subunit-resolved 76 analysis of ENaC architecture in native lysates. These tools establish a robust platform 77 for dissecting ENaC assembly, composition, and regulation directly in physiological 78 contexts. Because ENaC activity must be tuned to the distinct transport demands of 79 different epithelia, tissue-specific differences in subunit assembly and associations with 80 regulatory partners are likely to shape Na+ transport capacity and hormonal 81 responsiveness1-4, 39, 41-44. 82 83

84 85 Generation of the ENaCγ-VF mouse line 86 To enable detection and purification of native ENaC complexes, we designed a knock-in 87 mouse line in which the endogenous Scnn1g locus (encoding ENaCγ) was modified to 88 incorporate a C-terminal mVenusQ69M fluorophore, a 3C protease recognition site, and a 89 3xFLAG epitope50-52 (Fig. 1a). This multifunctional design enables direct fluorescence 90 detection, native-state affinity isolation, and protease-mediated elution of intact ENaC 91 assemblies. The ENaCγ-VF allele was generated at the Jackson Laboratory using 92 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 6 CRISPR/Cas9 to insert the tag in-frame at the endogenous Scnn1g stop codon53, 54. 93 Initial characterization was performed in heterozygous ENaCγ-VF+/- mice to assess 94 whether C-terminal tagging of γENaC perturbed channel expression, trafficking, or 95 subcellular localization in the presence of an unmodified endogenous allele. 96 Heterozygous ENaCγ-VF+/- mice exhibited normal appearance and growth, and 97 immunostaining of kidney sections confirmed correct apical localization of the tagged γ 98 subunit within the distal nephron (Fig. S1), consistent with preserved trafficking and 99 expression40. 100 101 A central requirement for using this model to study native ENaC is that the modified γ 102 subunit must independently support physiological channel function on its own. Because 103 Scnn1g knockout is perinatally lethal55, viability in the homozygous state provides a 104 stringent functional test of the tagged allele. Homozygous ENaCγ-VF/ENaCγ-VF mice 105 were viable into adulthood, indicating that the mVenus-3C-3xFLAG fusion does not 106 disrupt essential ENaC activity. These animals therefore served as the foundation for all 107 subsequent physiological, biochemical, and biophysical analyses. 108 109 Validation of the ENaCγ-VF mouse line 110 We validated the expression and localization of the tagged γ subunit in kidney tissue, 111 where ENaC plays a well-defined role in sodium reabsorption. Immunostaining showed 112 that mVenus-tagged γ was appropriately restricted to the distal nephron, consistent with 113 the known distribution of native ENaC40 (Fig. 1b, c). Blood electrolyte levels in ENaCγ-114 VF mice were within the normal physiological range (Fig. 2a). To assess whether the 115 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 7 tagged γ subunit supports normal channel function in vivo, we examined the response 116 of ENaCγ-VF mice to amiloride, a well-characterized ENaC inhibitor56. As expected, 117 pharmacological blockade increased the urinary Na⁺:K⁺ ratio, reflecting inhibition of 118 ENaC-mediated sodium reabsorption and potassium secretion in the distal nephron. 119 ENaCγ-VF mice exhibited an amiloride-induced increase in Na⁺:K⁺ ratio 120 indistinguishable from wild-type controls (Fig. 2b), indicating preserved ENaC function. 121 These findings demonstrate that homozygous ENaCγ-VF mice maintain normal 122 electrolyte handling and are phenotypically comparable to wild-type littermates under 123 basal conditions. 124 125 To complement these physiological measurements with a direct biochemical 126 assessment of channel pharmacology, we performed radioligand filter-binding assays 127 using tritiated [³H]-benzamil, a high-affinity ENaC antagonist57. Kidney membranes were 128 selected for these experiments because renal ENaC activity underlies the in vivo 129 amiloride challenge and provides a robust, well-defined tissue context in which ENaC-130 dependent sodium transport can be directly interrogated. Kidney membrane fractions 131 were isolated, incubated with increasing concentrations of [³H]-benzamil, and filtered to 132 separate receptor-bound from free radioligand. Specific benzamil binding was readily 133 detectable in both wild-type and ENaCγ-VF membranes (Fig. 2c). The two genotypes 134 exhibited comparable apparent dissociation constants (Kd = 13 nM for wild-type and 17 135 nM for ENaCγ-VF). These values indicate that the C-terminal mVenus-3C-3xFLAG tag 136 does not measurably alter benzamil affinity. The physiological and biochemical data 137 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 8 establish that the tagged γ subunit preserves native ENaC function and 138 pharmacological properties. 139 140 Detection and characterization of native ENaC complexes using FSEC and 141 SiMPull 142 The mVenus tag provides a sensitive fluorescence handle for tracking γ-containing 143 ENaC assemblies in tissue lysates using both ensemble and single-molecule 144 approaches (Fig. 3a). FSEC48 separates protein complexes by size under non-145 denaturing conditions and uses inline fluorescence to monitor tagged species as they 146 elute from a calibrated gel-filtration column. Originally developed for recombinant 147 membrane proteins, FSEC has proven effective for assessing complex formation, 148 sample homogeneity, and expression levels48, 58. Here, we extend this approach to 149 native tissues by using mVenus as an intrinsic reporter for γ-containing ENaC 150 complexes. 151 152 We applied FSEC to characterize native ENaC assemblies in lung, kidney, and colon 153 tissues from ENaCγ-VF mice. Lysates were fractionated on a Superose 6 Increase 154 column, and mVenus fluorescence was monitored across the elution profile (Fig. 3a). 155 Lung and kidney lysates displayed well-defined fluorescence peaks eluting between 11 156 and 16 mL, consistent with the expected size range of multimeric ENaC complexes (Fig. 157 3b). In contrast, colon lysates produced substantially lower signal in initial FSEC runs. 158 Free mVenus eluted at a later volume, allowing us to distinguish intact ENaCγ-159 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 9 containing complexes from unincorporated fluorophore and to verify sample integrity 160 prior to downstream analyses. 161 162 To complement ensemble analyses and resolve the ENaC composition at the single-163 complex level, we integrated SiMPull49. SiMPull combines immunoaffinity capture with 164 total internal reflection fluorescence (TIRF)59 microscopy to visualize individual protein 165 complexes under non-denaturing conditions. This approach is well suited for low-166 abundance membrane proteins such as ENaC, offering attomole-scale sensitivity60 167 while preserving native subunit interactions. For selective capture of ENaCγ-VF, we 168 employed a biotinylated anti-GFP/mVenus nanobody61 that binds to the mVenus 169 fluorophore and enables immobilization of γ-containing assemblies on PEG-passivated, 170 streptavidin-coated glass surfaces. 171 172 FSEC was used not only as an ensemble characterization but also as a preparative 173 step to ensure that single-molecule measurements interrogated intact complexes. 174 Fractions eluting before free mVenus, corresponding to high-molecular-weight γ-175 containing assemblies, were selected for SiMPull. Using this FSEC-guided workflow, 176 robust mVenus signals were detected in lung and kidney samples, confirming efficient 177 and specific capture of γ-containing complexes (Fig. 3c, d). For colon samples, where 178 FSEC indicated lower ENaC abundance, lysates were concentrated prior to SiMPull, 179 which enabled reliable single-molecule detection. 180 181 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 10 The combined use of FSEC and SiMPull provides a powerful, complementary platform 182 for characterizing native ENaC assemblies. FSEC offers a rapid assessment of complex 183 integrity and abundance, while SiMPull resolves individual complexes with single-184 molecule precision. These approaches establish a robust analytical platform for 185 studying ENaC composition and assembly across multiple epithelial tissues. The 186 observed tissue-dependent differences in ENaC abundance and apparent complex size 187 are consistent with known variations in baseline Na+ transport and ENaC dependence 188 across lung, kidney, and colon, and may contribute to how each epithelium matches 189 channel activity to its specific absorptive demands1-7, 40. 190 191 Dual-color FSEC using a fluorescent Fab against ENaCα 192 Having established that ENaCγ-VF provides a reliable fluorescence handle for tracking 193 γ-containing assemblies across tissues, we next asked whether this platform could be 194 extended to resolve the composition of native ENaC complexes at the subunit level. 195 Because the mVenus tag reports specifically on the γ subunit, the analyses above 196 define the behavior of γ-containing assemblies but do not directly address the 197 incorporation of other ENaC subunits into the same complexes. A key advantage of the 198 ENaCγ-VF allele is that it provides a stable biochemical anchor from which additional 199 subunits can be assessed. To expand the detection capabilities of the system and 200 enable subunit-resolved interrogation of native ENaC assemblies, we developed a 201 fluorescent Fab directed against the extracellular domain of ENaCα. 202 203 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 11 The parent monoclonal antibody, 7B1, was originally raised against human ENaCα35, 204 and sequence alignment revealed conservation between human and mouse α subunits 205 within the predicted epitope (Fig. 4a, b), supporting the feasibility of cross-species 206 recognition. To generate a monovalent, fluorescent reagent suitable for biochemical and 207 biophysical studies, we cloned the variable regions of the 7B1 heavy and light chains 208 into secretion-optimized expression constructs and fused the heavy chain C terminus to 209 the red fluorescent protein mScarlet-I62, yielding 7B1-mScarlet (Fig. 4c). The Fab was 210 secreted efficiently from HEK293 cells and isolated from conditioned media. 211 212 We first validated 7B1-mScarlet in a recombinant system expressing mouse ENaC 213 composed of wild-type α and β subunits and a mVenus-tagged γ subunit, recapitulating 214 the configuration of the ENaCγ-VF allele (Fig. S2a). Detergent-solubilized lysates were 215 incubated with 7B1-mScarlet and analyzed by FSEC. mVenus and mScarlet 216 fluorescence co-eluted in a single peak at the expected position for trimeric ENaC, 217 whereas control samples lacking 7B1-mScarlet showed no mScarlet signal within this 218 elution window (Fig. S2b). These results indicate that 7B1-mScarlet binds specifically to 219 folded ENaC complexes containing the α subunit. To assess whether 7B1 recognizes α 220 across different maturation states, we performed surface biotinylation of cells 221 expressing recombinant mouse ENaC, lysed them, purified ENaC via the γ tag, and 222 probed the resulting surface and intracellular fractions with 7B1-mScarlet (Fig. S2c); 223 robust signal was detected in both biotinylated and non-biotinylated pools, indicating 224 that the Fab can bind α in multiple maturation states rather than a single conformational 225 end point (Fig. S2d). 226 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 12 227 We next asked whether this Fab could detect ENaCα within native γ-containing 228 complexes isolated from tissue. Lung lysates from ENaCγ-VF mice were enriched for γ-229 containing assemblies by affinity capture through the C-terminal FLAG tag and eluted 230 under non-denaturing conditions. Addition of 7B1-mScarlet to the eluted material 231 followed by FSEC analysis revealed co-elution of mVenus and mScarlet fluorescence 232 (Fig. 4d-f), demonstrating that α and γ subunits are incorporated into the same native 233 complexes. Strikingly, while mVenus fluorescence extended across a broader range (11-234 16 mL), the 7B1-mScarlet signal was confined to the 11-13 mL window. These data 235 establish dual-color FSEC as a non-denaturing approach for resolving the subunit 236 composition of native γ-containing ENaC assemblies. The distinct behavior indicates 237 that α and γ subunits partition into different subsets of γ-containing assemblies and 238 highlights heterogeneity within the γ-containing population. The observations motivated 239 further investigation into whether γ-containing assemblies of differing apparent sizes 240 engage distinct regulatory protein complexes in vivo. 241 242 ENaCγ-containing complexes associate with regulatory proteins in vivo 243 The heterogeneity revealed by dual-color FSEC raised the possibility that g-containing 244 assemblies of different apparent sizes represent distinct molecular states. The presence 245 of both a-containing complexes and broader g-containing assemblies is consistent with 246 a model in which these complexes correspond to distinct maturation or stability 247 checkpoints that tune ENaC expression14-25, 39-44. In this context, the 7B1-positive 248 population represents α-containing complexes enriched within a narrower, higher 249 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 13 molecular weight fraction, whereas γ-based fluorescence reports a continuum of γ-250 containing assemblies likely spanning multiple stages of folding, assembly, and 251 regulatory interactions. To examine this possibility and extend our analysis beyond 252 ENaC subunits alone, we turned to mass spectrometry to identify proteins that co-purify 253 with γ-containing assemblies. 254 255 Large-scale affinity purification was performed from lung, kidney, and colon tissues of 256 ENaCγ-VF mice using anti-FLAG enrichment followed by 3C protease cleavage to 257 gently release native complexes under non-denaturing conditions (Fig. 5a). Fractions 258 corresponding to the mVenus fluorescence peak were collected and subjected to mass 259 spectrometry analysis (Fig. 5b). As expected, ENaCγ co-purified with ENaCα and β 260 subunits (Fig. 5c), consistent with our dual-color FSEC data demonstrating α-γ co-261 assembly (Fig. 4f). Recovery of all three subunits confirms the integrity of the purified 262 complexes and demonstrates that the ENaCγ-VF tag preserves native channel 263 assembly. 264 265 In addition to the core ENaC subunits, mass spectrometry revealed several regulatory 266 proteins that associate with γ-containing assemblies. Notably, the E3 ubiquitin ligase 267 Nedd4-243 was detected across multiple tissues. Nedd4-2 is a well-established regulator 268 of ENaC surface expression and turnover, mediating ubiquitin-dependent internalization 269 and degradation. Its presence suggests that a subset of γ-containing complexes 270 engages components of the ubiquitin regulatory pathway. We also detected the ER 271 chaperone calnexin, consistent with its established role in the folding and quality control 272 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 14 of multi-subunit membrane proteins63-65, including ENaC. Its presence suggests that 273 another subset of γ-containing complexes engages components of the protein quality -274 control pathway. These associations support a model in which γ participates both in fully 275 assembled ENaC channels and in regulatory-associated forms, providing a molecular 276 basis for the broader elution profile observed by FSEC. 277 278 To evaluate the specificity of these interactions, we performed identical affinity 279 purifications from wild-type tissues lacking the mVenus-3C-3xFLAG tag (Fig. S3). 280 Neither Nedd4-2 nor calnexin was detected in these control samples, indicating that 281 their presence in ENaCγ-VF purifications reflects specific co-purification with γ-282 containing complexes rather than nonspecific retention on the affinity resin. These 283 findings broaden the scope of our analysis from subunit composition to the regulatory 284 environment surrounding native ENaC. The ENaCγ-VF allele enables the isolation of 285 both assembled αβγ channels, as well as γ-associated intermediary or regulatory 286 complexes, linking biochemical heterogeneity observed by FSEC to defined molecular 287 pathways that govern ENaC maturation, quality control, and turnover in vivo. 288 289

290 This study introduces the ENaCγ-VF knock-in mouse line as a physiologically faithful 291 platform for dissecting the composition, assembly, and regulatory environment of native 292 ENaC complexes across epithelial tissues. By combining endogenous fluorescent 293 tagging with orthogonal biochemical and single-molecule approaches, this system 294 overcomes longstanding challenges posed by ENaC’s low abundance, membrane 295 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 15 localization, and complex subunit stoichiometry. Preservation of normal physiology in 296 homozygous ENaCγ-VF mice demonstrates that the C-terminal mVenus-3C-3xFLAG 297 fusion maintains native channel function, providing confidence that downstream 298 biochemical and structural analyses reflect bona fide ENaC biology. 299 300 A notable strength of the ENaCγ-VF allele is its broad compatibility with both advanced 301 and routine biochemical and imaging workflows. Here, we demonstrate that the mVenus 302 tag enables sensitive detection by ensemble fluorescence, FSEC, confocal imaging, 303 and TIRF microscopy. More generally, mVenus and FLAG tags also support robust 304 detection by standard immunoblotting using well-validated commercial antibodies, 305 making the mouse line readily compatible with widely-used biochemical analyses. In 306 parallel, the C-terminal 3xFLAG epitope supports affinity purification followed by native-307 state elution using 3C protease. These features allow intact ENaC assemblies to be 308 isolated without disrupting subunit composition or regulatory interactions. Thus, the 309 ENaCγ-VF mouse provides a practical and adaptable foundation for examining ENaC at 310 molecular, cellular, and tissue scales. 311 312 FSEC and SiMPull analyses reveal substantial heterogeneity among γ-containing ENaC 313 complexes across lung, kidney, and colon. FSEC profiles identify both well-defined 314 trimeric assemblies and broader, higher-molecular-weight populations, while SiMPull 315 confirms that γ-containing complexes can be isolated and visualized at the single-316 molecule level under non-denaturing conditions. These findings indicate that ENaCγ 317 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 16 participates in multiple molecular states in vivo and underscore the utility of the ENaCγ-318 VF system for resolving this complexity directly. 319 320 To distinguish fully assembled channels from intermediate or regulatory states, we 321 developed 7B1-mScarlet, a fluorescent Fab that recognizes ENaCα under mild 322 conditions. Dual-color FSEC showed that α-containing complexes elute within a 323 narrower molecular range than γ-containing assemblies, suggesting preferential 324 incorporation of α into a more restricted subset of αβγ channels. Validation in 325 recombinant and native systems confirmed that 7B1-mScarlet selectively binds folded α 326 within intact ENaC complexes. 327 328 Within this context, γ occupies a central position in the ENaC assembly landscape. 329 Unlike α, which appears to be selectively incorporated into a narrower population of 330 complexes, γ persists across multiple assembly and regulatory states, positioning it as a 331 molecular hub for ENaC biogenesis and turnover. The broad elution range detected by 332 FSEC, the presence of γ in both trimeric channels and regulatory-associated species, 333 and its interaction with chaperones and ubiquitin ligases collectively suggest that γ 334 participates in multiple checkpoints along the channel’s biogenesis, quality control, and 335 turnover pathways. In contrast, α appears in a more restricted set of molecular contexts, 336 consistent with its rate-limiting role in channel assembly and preferential incorporation 337 into fully assembled αβγ complexes. The sharp, 7B1-positive peak therefore most likely 338 reflects mature, fully assembled αβγ channels, whereas the broader γ profile 339 encompasses assembly intermediates and regulator -engaged species. This 340 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 17 interpretation is in line with quantitative biochemical studies showing an excess of β 341 and γ over α subunits in native epithelia, implying that only a subset of γ participates in 342 fully assembled channels at any given time 32. These observations align with long-343 standing physiological models in which differences in subunit abundance govern ENaC 344 assembly efficiency, but here are supported by direct biochemical analysis of intact 345 native complexes. These findings indicate that native ENaC exists not as a single, static 346 entity but as an ensemble of molecular species that differ in subunit composition, 347 assembly state, and regulatory interactions. Decades of physiological and 348 electrophysiological studies inferred such heterogeneity indirectly, but the lack of tools 349 to visualize and isolate native complexes obscured the underlying molecular 350 architecture66. The ENaCγ-VF model, particularly when combined with subunit-specific 351 probes such as 7B1-mScarlet and native-state purification, begins to resolve this 352 complexity with a level of precision that was previously inaccessible. 353 354 Mass spectrometry further supports this view by identifying regulatory factors that co-355 purify with γ-containing assemblies. In addition to α and β subunits, we detected Nedd4-356 2 and calnexin, key regulators of ENaC maturation, trafficking, and turnover. Their 357 selective recovery in ENaCγ-VF purifications, demonstrates that affinity isolation via the 358 γ subunit specifically enriches bona fide γ-associated ENaC complexes rather than 359 nonspecific background proteins. The identification of Nedd4-2 and calnexin within 360 these purifications further indicates that a subset of γ-containing assemblies engages 361 quality-control machinery and regulatory pathways in vivo. This association provides a 362 molecular explanation for the broader elution profile of γ-containing species and 363 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 18 highlight the dynamic equilibrium between mature channels, assembly intermediates, 364 and regulatory states in vivo. Taken together with the preserved physiology of ENaCγ-365 VF mice and the specific recovery of known ENaC regulators, these observations 366 argue that the γ-containing species primarily reflect native assembly and quality -control 367 states rather than artifacts of the γ-tag. 368 369 More broadly, the ENaCγ-VF platform enables a transition from indirect inference to 370 direct molecular definition of native ENaC assemblies. By enabling isolation, 371 visualization, and quantification of intact ENaC complexes under non-denaturing 372 conditions, this system provides a foundation for linking subunit composition and 373 regulatory state to tissue-specific ENaC function. As complementary tools for detecting 374 additional ENaC subunits, particularly β, and for structural analysis of native complexes 375 continue to emerge, the ENaCγ-VF model will serve as a foundational resource for 376 defining the molecular principles that govern ENaC regulation across epithelial systems. 377 In future work, this platform can be applied to examine how disease states remodel 378 ENaC assembly and regulatory interactions, linking molecular architecture to disordered 379 Na⁺ handling in conditions such as salt-sensitive hypertension, cystic fibrosis, and 380 pseudohypoaldosteronism. 381

382 Generation of ENaCγ-VF mice 383 The ENaCγ-VF knock-in mouse line was generated by the Jackson Laboratory using 384 CRISPR/Cas9-mediated genome editing to insert a sequence encoding mVenus, a 3C 385 protease cleavage site, and a 3xFLAG tag at the C-terminus of the endogenous Scnn1g 386 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 19 gene. Heterozygous animals (ENaCγ-VF/+) were initially shipped to our lab for 387 validation. Following phenotypic and localization analysis (see Results), the Jackson 388 Laboratory provided homozygous (ENaCγ-VF/ENaCγ-VF) mice, which were also viable 389 and showed normal ENaCγ localization. Both heterozygous and homozygous mice are 390 maintained under standard breeding protocols. To ensure continued integrity of the 391 tagged allele, we routinely verify the line using genotyping services with primers 392 designed to detect the presence of the mVenus-3xFLAG insertion. All animal 393 procedures were conducted in accordance with protocols approved by the IACUC. 394 Immunofluorescence Microscopy 395 Mice were anesthetized with a ketamine-xylazine-acepromazine cocktail (50:5:0.5 396 mg/kg). The kidneys were perfusion fixed by retrograde abdominal aortic perfusion of 397 3% paraformaldehyde in PBS (pH 7.4). After perfusion, the kidneys were removed, 398 dissected, and cryopreserved in 800 mOsm/L sucrose in PBS overnight before being 399 embedded in Tissue-Tek Optimal Cutting Temperature compound (Sakura Finetek, 400 Torrance, CA). Slides were prepared by cutting 5 mm sections and stored at -80°C until 401 use. For imaging, slides were incubated with 0.5% Triton X-100 in PBS for 30 minutes, 402 blocked with 5% milk in PBS for 30 minutes, followed by incubation with primary 403 antibody, diluted in blocking buffer, for 1 hour at room temperature or overnight at 4°C. 404 Sections were then washed with PBS three times and incubated with fluorescent dye-405 conjugated secondary antibody, diluted in blocking buffer, for 1 hour at room 406 temperature. Sections were washed with PBS three times and stained with 4’,6-407 diamidino-2-phenylindole before being mounted with ProLong Diamond Antifade 408 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 20 Mountant (ThermoFisher Scientific, Carlsbad, CA). Images were captured using a 409 KEYENCE BZ-X800 microscope (Itasca, IL). 410 411 Amiloride treatment and urine analysis 412 Mice were injected intraperitoneally with vehicle (0.9% saline) and then placed in 413 metabolic cages for a 6-hour urine collection. Five days later, the same animals 414 received intraperitoneal injection of amiloride hydrochloride (40 μg per 25 g body 415 weight), followed by another 6-hour urine collection. Sodium and potassium 416 concentrations were measured by flame photometry, and the urinary Na⁺:K⁺ ratio was 417 used to assess ENaC function. 418 Filter binding assay 419 Kidney tissue was dissected from adult wild-type or ENaCγ-VF mice, rinsed in ice-cold 420 tris buffered saline (TBS, 20mM Tris-HCl pH 7.6, 200mM NaCl), stored in TBS with 421 protease inihibitors (Thermo Scientific™ Pierce protease inhibitor tablets, ThermoFisher 422 Scientific) and flash-frozen in liquid nitrogen. For membrane preparation, frozen kidneys 423 were pulverized using a liquid nitrogen-cooled mortar and pestle until a fine powder was 424 obtained. Tissue was then homogenized in homogenization buffer (50mM phosphate 425 buffer, pH 7.5) using a Dounce homogenizer. The homogenate was centrifuged at 426 1,800g for 10 min at 4 °C to remove nuclei and cellular debris. The resulting 427 supernatant was further centrifuged at 100,000g for 60 min at 4 °C to pellet membrane 428 fractions. Membrane pellets were resuspended in binding buffer (50mM phosphate 429 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 21 buffer, pH 7.5). Equilibrium ligand binding was assessed using tritiated benzamil ([³H]-430 benzamil; Moravek). Membrane suspensions (typically 10 mg) were incubated with 431 increasing concentrations of [³H]-benzamil (0.5-300 nM) in a final volume of 500 µL 432 binding buffer. Incubations were carried out at 22 °C for 2 hours, a duration sufficient to 433 reach equilibrium under these conditions. Nonspecific binding was determined in 434 parallel samples containing 100 µM unlabeled phenamil (Phenamil mesylate, Tocris 435 Bioscience). Binding reactions were terminated by rapid vacuum filtration onto 436 presoaked glass microfiber filters (GF/B 25mm, Whatman) using a 12-well filtration 437 manifold (Millipore). Filters were prewashed with 2ml ice-cold binding buffer and treated 438 with 0.3% polyethyleneimine (PEI, MW 25K, Polysciences) to reduce nonspecific 439 radioligand adsorption. Incubated samples were applied and each well was washed 440 immediately with 2 x 5 mL ice-cold binding buffer. Filters were dried by vacuum, 441 transferred to scintillation vials, extracted in 5 mL scintillation cocktail (Ultima Gold), and 442 radioactivity was quantified by liquid scintillation counting (Beckman Coulter LS6500). 443 Specific binding was calculated as total minus nonspecific counts per minute (CPM). 444 Equilibrium binding curves were fitted to a one-site binding model using nonlinear 445 regression in GraphPad Prism: 446 𝑌 = 𝐵max 𝑋 𝐾! + 𝑋 448 447 where Y is specific binding, X is the free ligand concentration, Bₘₐₓ is maximal binding, 449 and Kd is the apparent dissociation constant. Data from ENaCγ-VF and wild-type 450 samples were fitted independently, and similar Kd values were interpreted as evidence 451 that the γ-tag does not perturb benzamil affinity. 452 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 22 Tissue preparation and lysate generation for FSEC analysis 453 Tissues were harvested from ENaCγ-VF mice. For each FSEC experiment, one lung (all 454 lobes), one kidney, and two colons were collected, rinsed in cold TBS, and weighed. 455 Each tissue was homogenized in solubilization buffer containing 100 mM Tris-HCl (pH 456 7.6), 200 mM NaCl, 1% n-dodecyl-β-D-maltoside (DDM, Anagrade, Anatrace), protease 457 inhibitors and Pefabloc® SC (Millipore Sigma). The homogenization volume was 458 standardized to 750 µL per 100 mg of tissue. Samples were solubilized at 4 °C for 60 459 minutes with gentle rotation, then clarified by centrifugation at 100,000g twice for 40 460 minutes each. Supernatants were concentrated using a 100kDa centrifugal concentrator 461 to a final volume of 110 - 480 µL. Concentrated samples were then centrifuged again at 462 100,000g for 10 minutes to remove aggregates. The final cleared supernatant was 463 injected onto a Superose 6 Increase column (Cytiva) for FSEC analysis (Waters 464 HPLC)48. Fractions were collected and analyzed based on mVenus fluorescence. 465 SiMPull Assays 466 Glass coverslips were cleaned and PEGylated using standard silanization procedures49, 467 then functionalized sequentially with streptavidin and a biotinylated anti-GFP/mVenus 468 nanobody67. One experimental channel included the nanobody to capture fluorescently 469 tagged ENaC subunits, while a parallel channel without nanobody was used to quantify 470

signal. 471 Streptavidin was applied at a concentration of 250 µg/mL and incubated for 5 minutes at 472 room temperature, followed by a wash with the assay buffer (20 mM Tris-HCl (pH 7.6), 473 200 mM NaCl, 0.025% n-dodecyl-β-D-maltoside (DDM)) containing 200 µg/mL BSA. 474 Biotinylated nanobody was then applied at 3 µg/mL and incubated for 10 minutes, 475 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 23 followed again by a wash to remove unbound reagent. Clarified cell lysates (prepared 476 as for FSEC analysis) containing fluorescently tagged ENaC subunits were then applied 477 and incubated for 10 minutes at room temperature, followed by a final wash before 478 imaging. Chambers were imaged on a Leica DMi8 TIRF microscope with a 100x oil-479 immersion objective. Images were captured using an Andor iXon Ultra 888 back-480 illuminated EMCCD camera with a 133 x 133 µm imaging area and 130 nm pixel size. 481 For counting fluorophore spots, images were acquired using the excitation wavelength 482 488 nm. Spot detection and localization were performed using the ComDet plugin in 483 ImageJ (FIJI). Molecule positions were analyzed within the center area of the field of 484 view (512x512 px). The approximate particle size was set at 5 pixels. The spot intensity 485 threshold (in SD) was set to 3. Data were statistically analyzed using GraphPad Prism. 486 487 Affinity Isolation of Native ENaC Complexes for FSEC and Mass Spectrometry 488 Tissue preparation for this assay was performed using the same protocol as described 489 for FSEC analysis, with the exception that digitonin was used as the detergent for 490 solubilization instead of DDM. Briefly, lungs, kidneys, and colons were collected from 5 491 mice, and homogenized in solubilization buffer containing 100 mM Tris-HCl pH 7.6, 200 492 mM NaCl, 5mM EDTA, 1% digitonin (High purity, Millipore Sigma), protease inhibitors 493 and Pefabloc. Clarified lysates were incubated with 50 µL of anti-FLAG M2 magnetic 494 beads (Sigma-Aldrich) overnight at 4 °C with gentle rotation. Beads were washed 10 495 times with 15 ml wash buffer (20 mM Tris-HCl pH 7.6, 200 mM NaCl, 0.1% digitonin), 496 and proteins were eluted by incubating with 25 µg of PreScission Proteases (GenScript) 497 for 6 hours at 4 °C. The eluted material, in a total volume of 400 uL, was concentrated 498 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 24 using centrifugal filters prior to injection onto a Superose 6 Increase 10/300 column 499 (Cytiva) equilibrated in FSEC buffer (20 mM Tris-HCl pH 7.6, 200 mM NaCl, 0.1% 500 digitonin). Fluorescence was monitored using an inline fluorimeter (Shimadzu RF-20A) 501 to track mVenus-tagged ENaCγ. Fractions corresponding to the fluorescent signal were 502 collected, pooled, and concentrated again for downstream mass spectrometry analysis. 503 504 7B1-mScarlet Fab Production 505 DNA sequences encoding the heavy and light chains of the Fab fragment of the anti-506 ENaCα monoclonal antibody 7B1 were cloned into separate pEG BacMam expression 507 plasmids68. The heavy chain construct was engineered to include mScarlet-I at the C-508 terminus, followed by a Strep-tag II for affinity purification. Both constructs included an 509 N-terminal leader sequence (MGWSCIILFLVATATGVHS) that allows for secretion of the 510 Fab into the media. First passage of mBaculovirus was generated from each plasmid 511 using Sf9 insect cells cultured at a density of 0.5x10⁶ cells/mL at 27 °C for 5 days. P1 512 virus was used to reinfect Sf9 insect cells cultured at a density of 1x10⁶ cells/mL at 513 27 °C for 4 days. After incubation, cells were pelleted by centrifugation at 1,800g for 20 514 minutes, and the supernatant containing the virus was harvested and filtered through a 515 0.22 µm filter. The resulting virus was used to infect suspension-adapted HEK293 cells 516 at a density of 3x10⁶ cells/mL and incubated at 37 °C for 8 hours. After 8 hours, 10mM 517 sodium butyrate was added and the cells were moved to a 30 °C incubator. Cells were 518 cultured at 30 °C for 96 hours, after which they were spun down and the culture 519 supernatant was collected and filtered again using a 0.22 µm filter. The clarified media 520 with BioLock (1.6ml/L of FreeStyle™ 293, IBA Lifesciences) was loaded onto a 10 mL 521 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 25 Strep-Tactin affinity resin (5ml/1L of media) (Cytiva) pre-equilibrated with wash buffer 522 (20mM Tris pH 7.6, 200mM NaCl and 0.025% n-dodecyl-β-D-maltoside (DDM)). The 523 column was washed with 20 column volumes of wash buffer, and bound 7B1-mScarlet 524 Fab was eluted using desthiobiotin-containing wash buffer. Desthiobiotin (IBA 525 Lifesciences) was subsequently removed using a PD-10 desalting column (Cytiva), and 526 the Fab was eluted in wash buffer. Purity and labeling of the Fab were confirmed by 527 SDS-PAGE and FSEC. 528 529 Cell-surface biotinylation and validation of 7B1-mScarlet binding to recombinant 530 ENaC 531 To evaluate binding of the 7B1-mScarlet Fab to recombinant mouse ENaC, we 532 expressed a mouse ENaC construct composed of the α (Uniprot ID Q61180), β (Uniprot 533 ID Q9WU38), and γ (Uniprot ID Q9WU39) subunits, with the γ subunit bearing the C-534 terminal mVenus tag. Cells were analyzed under both total and plasma membrane-535 enriched conditions. For total protein analysis, cells were solubilized directly36, 37, 536 clarified by centrifugation, and the resulting lysates were incubated with 7B1-mScarlet 537 prior to downstream analysis. To specifically assess plasma membrane-expressed 538 ENaC, cell-surface proteins were labeled using a membrane-impermeant biotinylation 539 reagent according to the manufacturer’s instructions (Pierce™ Cell surface biotinylation 540 & Isolation Kit, ThermoFisher Scientific). Briefly, cells expressing recombinant mouse 541 ENaC were harvested and incubated with biotin (EZ-Link Sulfo-NHS-SS-Biotin) to 542 selectively label surface-exposed proteins. Excess biotin was quenched and removed 543 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 26 by washing, after which cells were either flash-frozen for storage at -80 °C or processed 544 immediately for solubilization. 545 546 Cells were lysed in buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 20mM 547 DDM, 3mM CHS, and protease inhibitors. Lysates were clarified by centrifugation, and 548 the supernatant was incubated with GFP nanobody resin (GNB). Following binding, the 549 resin was washed with 20mM Tris (pH7.6), 150mM NaCl, 0.5mM DDM, 75uM CHS 550 without and with 5mM CaCl2. Both biotinylated and non-biotinylated fractions of the 551 protein were eluted by thrombin (30ug/ml of resin, Human alpha-thrombin, Prolytix) 552 cleavage. The thrombin eluted sample was passed through the Strep-Tactin resin to 553 capture biotinylated, plasma membrane-expressed proteins. The unbound flow-554 through, representing the intracellular protein fraction, was collected. Following 555 fractionation, 7B1-mScarlet was added to the flow-through fraction. Biotinylated proteins 556 bound to the Strep-Tactin resin were subsequently eluted using excess biotin (10x 557 Buffer BXT, IBA Lifesciences), and 7B1-mScarlet was added to the eluted plasma 558 membrane fraction. Both intracellular (flow-through) and plasma membrane (biotin-559 eluted) fractions were analyzed by HPLC, with fluorescence monitored in 560 the mScarlet channel. 561 Data Availability Statement 562 All data in this manuscript are available from the corresponding author upon request. 563 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 27

564 We thank members of the Gouaux laboratory, especially Eric Gouaux, April Goehring, 565 and Natalie Sheldon, for their generous guidance and support as we established 566 animal-based experiments and native-state biochemical workflows. We are grateful to 567 Ashok Reddy and the staff of the OHSU Proteomics Shared Resource for mass 568 spectrometry analyses and technical expertise. We thank the OHSU Department of 569 Comparative Medicine for assistance with animal care and colony management. The 570 ENaCγ-VF mouse line was generated by The Jackson Laboratory. Funding for this 571 project was provided by institutional support from the Vollum Institute and by NIH grant 572 R01GM138862 to I.B, R01DK132066 to J.A.M, and R01s DK133220 and DK51496 to 573 D.E. 574 575 Author Contributions 576 A.B. maintained the mouse colony, harvested mouse tissues, and performed FSEC, 577 SiMPull, and purification of native ENaC complexes. J.C. performed the amiloride 578 challenge experiments. X.-T.S. conducted confocal imaging of heterozygous and 579 homozygous mice. R.B.M. maintained the mouse colony and harvested mouse tissues. 580 J.A.M. and D.H.E. supervised the physiology experiments. I.B. conceived and 581 supervised the study. All authors contributed to manuscript preparation. 582 583 Declaration of interests 584 The authors declare no competing interests. 585 586 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 28

587 588 1. Hamm LL, Feng Z, Hering-Smith KS. Regulation of sodium transport by ENaC in 589 the kidney. Curr Opin Nephrol Hypertens. 2010;19(1):98-105. doi: 590 10.1097/MNH.0b013e328332bda4. PubMed PMID: 19996890; PMCID: PMC2895494. 591 2. Matalon S, Bartoszewski R, Collawn JF. Role of epithelial sodium channels in the 592 regulation of lung fluid homeostasis. Am J Physiol Lung Cell Mol Physiol. 593 2015;309(11):L1229-38. Epub 2015/10/04. doi: 10.1152/ajplung.00319.2015. PubMed 594 PMID: 26432872; PMCID: PMC4669342. 595 3. Rossier BC, Pradervand S, Schild L, Hummler E. Epithelial sodium channel and 596 the control of sodium balance: interaction between genetic and environmental factors. 597 Annu Rev Physiol. 2002;64:877-97. doi: 10.1146/annurev.physiol.64.082101.143243. 598 PubMed PMID: 11826291. 599 4. Hanukoglu I, Hanukoglu A. Epithelial sodium channel (ENaC) family: Phylogeny, 600 structure-function, tissue distribution, and associated inherited diseases. Gene. 601 2016;579(2):95-132. Epub 2016/01/17. doi: 10.1016/j.gene.2015.12.061. PubMed 602 PMID: 26772908; PMCID: PMC4756657. 603 5. Matthay MA, Folkesson HG, Verkman AS. Salt and water transport across 604 alveolar and distal airway epithelia in the adult lung. Am J Physiol. 1996;270(4 Pt 605 1):L487-503. doi: 10.1152/ajplung.1996.270.4.L487. PubMed PMID: 8928808. 606 6. Gaillard D, Hinnrasky J, Coscoy S, Hofman P, Matthay MA, Puchelle E, Barbry P. 607 Early expression of beta- and gamma-subunits of epithelial sodium channel during 608 human airway development. Am J Physiol Lung Cell Mol Physiol. 2000;278(1):L177-84. 609 doi: 10.1152/ajplung.2000.278.1.L177. PubMed PMID: 10645905. 610 7. Malsure S, Wang Q, Charles RP, Sergi C, Perrier R, Christensen BM, Maillard M, 611 Rossier BC, Hummler E. Colon-specific deletion of epithelial sodium channel causes 612 sodium loss and aldosterone resistance. J Am Soc Nephrol. 2014;25(7):1453-64. Epub 613 20140130. doi: 10.1681/ASN.2013090936. PubMed PMID: 24480829; PMCID: 614 PMC4073440. 615 8. Mall MA. ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR 616 modulator therapies. Eur Respir J. 2020;56(6). Epub 20201224. doi: 617 10.1183/13993003.00946-2020. PubMed PMID: 32732328; PMCID: PMC7758539. 618 9. Chang SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild 619 L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, Lifton RP. Mutations in subunits 620 of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, 621 pseudohypoaldosteronism type 1. Nat Genet. 1996;12(3):248-53. Epub 1996/03/01. doi: 622 10.1038/ng0396-248. PubMed PMID: 8589714. 623 10. Kerem E, Bistritzer T, Hanukoglu A, Hofmann T, Zhou Z, Bennett W, MacLaughlin 624 E, Barker P, Nash M, Quittell L, Boucher R, Knowles MR. Pulmonary epithelial sodium-625 channel dysfunction and excess airway liquid in pseudohypoaldosteronism. N Engl J 626 Med. 1999;341(3):156-62. Epub 1999/07/15. doi: 10.1056/NEJM199907153410304. 627 PubMed PMID: 10403853. 628 11. Saxena A, Hanukoglu I, Saxena D, Thompson RJ, Gardiner RM, Hanukoglu A. 629 Novel mutations responsible for autosomal recessive multisystem 630 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 29 pseudohypoaldosteronism and sequence variants in epithelial sodium channel alpha-, 631 beta-, and gamma-subunit genes. J Clin Endocrinol Metab. 2002;87(7):3344-50. Epub 632 2002/07/11. doi: 10.1210/jcem.87.7.8674. PubMed PMID: 12107247. 633 12. Hummler E, Barker P, Talbot C, Wang Q, Verdumo C, Grubb B, Gatzy J, Burnier 634 M, Horisberger JD, Beermann F, Boucher R, Rossier BC. A mouse model for the renal 635 salt-wasting syndrome pseudohypoaldosteronism. Proc Natl Acad Sci U S A. 636 1997;94(21):11710-5. doi: 10.1073/pnas.94.21.11710. PubMed PMID: 9326675; 637 PMCID: PMC23605. 638 13. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, 639 Rossier BC. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-640 deficient mice. Nat Genet. 1996;12(3):325-8. Epub 1996/03/01. doi: 10.1038/ng0396-641 325. PubMed PMID: 8589728. 642 14. Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams 643 C, Rossier BC, Lifton RP. A de novo missense mutation of the beta subunit of the 644 epithelial sodium channel causes hypertension and Liddle syndrome, identifying a 645 proline-rich segment critical for regulation of channel activity1995. PubMed PMID: 646 13006205075825840505related:eVF7TJtSf7QJ. 647 15. Bubien JK, Ismailov, II, Berdiev BK, Cornwell T, Lifton RP, Fuller CM, Achard JM, 648 Benos DJ, Warnock DG. Liddle's disease: abnormal regulation of amiloride-sensitive 649 Na+ channels by beta-subunit mutation. Am J Physiol. 1996;270(1 Pt 1):C208-13. Epub 650 1996/01/01. doi: 10.1152/ajpcell.1996.270.1.C208. PubMed PMID: 8772446. 651 16. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, 652 Schambelan M, Gill JR, Ulick S, Milora RV, Findling JW. Liddle's syndrome: 653 heritable human hypertension caused by mutations in the beta subunit of the epithelial 654 sodium channel. Cell. 1994;79(3):407-14. PubMed PMID: 7954808. 655 17. Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB, 656 Welsh MJ. Mechanism by which Liddle's syndrome mutations increase activity of a 657 human epithelial Na+ channel. Cell. 1995;83(6):969-78. doi: 10.1016/0092-658 8674(95)90212-0. PubMed PMID: 8521520. 659 18. Schild L, Lu Y, Gautschi I, Schneeberger E, al e. Identification of a PY motif in the 660 epithelial Na channel subunits as a target sequence for mutations causing channel 661 activation found in Liddle syndrome. The EMBO …. 1996. PubMed PMID: C6CE859B-662 6F7D-4304-900D-D9D35CAA7367. 663 19. Warnock DG, Bubien JK. Liddle syndrome: clinical and cellular abnormalities. 664 Hosp Pract (Off Ed). 1994;29(7):95-8, 104-5. Epub 1994/07/15. PubMed PMID: 665 8027210. 666 20. Van Huysse JW, Amin MS, Yang B, Leenen FHH. Salt-induced hypertension in a 667 mouse model of Liddle syndrome is mediated by epithelial sodium channels in the brain. 668 Hypertension. 2012;60(3):691-6. doi: 10.1161/HYPERTENSIONAHA.112.193045. 669 PubMed PMID: 22802227; PMCID: PMC3514876. 670 21. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa 671 C, Iwasaki T, Rossier B, Lifton RP. Hypertension caused by a truncated epithelial 672 sodium channel γ subunit: genetic heterogeneity of Liddle syndrome. Nature Genetics. 673 1995;11(1):76-82. doi: 10.1038/ng0995-76. PubMed PMID: 674 3059133470290364987related:O0oq6a45dCoJ. 675 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 30 22. Sun Y, Zhang J-n, Zhao D, Wang Q-s, Gu Y-c, Ma H-p, Zhang Z-r. Role of the 676 epithelial sodium channel in salt-sensitive hypertension. Acta Pharmacologica Sinica. 677 2011;32(6):789-97. doi: 10.1038/aps.2011.72. 678 23. Pitzer AL, Van Beusecum JP, Kleyman TR, Kirabo A. ENaC in Salt-Sensitive 679 Hypertension: Kidney and Beyond. Curr Hypertens Rep. 2020;22(9):69. Epub 680 20200827. doi: 10.1007/s11906-020-01067-9. PubMed PMID: 32852643; PMCID: 681 PMC7452925. 682 24. Takahashi H, Yoshika M, Komiyama Y, Nishimura M. The central mechanism 683 underlying hypertension: a review of the roles of sodium ions, epithelial sodium 684 channels, the renin–angiotensin–aldosterone system, oxidative stress and 685 endogenous digitalis in the brain. Hypertension research : official journal of the 686 Japanese Society of Hypertension. 2011;34(11):1147-60. doi: 10.1038/hr.2011.105. 687 PubMed PMID: 21814209. 688 25. Gudmundsdottir H, Hoieggen A, Stenehjem A, Waldum B, Os I. Hypertension in 689 women: latest findings and clinical implications. Ther Adv Chronic Dis. 2012;3(3):137-690 46. doi: 10.1177/2040622312438935. PubMed PMID: 23251774; PMCID: 691 PMC3513905. 692 26. Lim SS, Vos T, Flaxman AD, Danaei G, Shibuya K, Adair-Rohani H, Amann M, 693 Anderson HR, Andrews KG, Aryee M, Atkinson C, Bacchus LJ, Bahalim AN, 694 Balakrishnan K, Balmes J, Barker-Collo S, Baxter A, Bell ML, Blore JD, Blyth F, Bonner 695 C, Borges G, Bourne R, Boussinesq M, Brauer M, Brooks P, Bruce NG, Brunekreef B, 696 Bryan-Hancock C, Bucello C, Buchbinder R, Bull F, Burnett RT, Byers TE, Calabria B, 697 Carapetis J, Carnahan E, Chafe Z, Charlson F, Chen H, Chen JS, Cheng AT-A, Child 698 JC, Cohen A, Colson KE, Cowie BC, Darby S, Darling S, Davis A, Degenhardt L, 699 Dentener F, Des Jarlais DC, Devries K, Dherani M, Ding EL, Dorsey ER, Driscoll T, 700 Edmond K, Ali SE, Engell RE, Erwin PJ, Fahimi S, Falder G, Farzadfar F, Ferrari A, 701 Finucane MM, Flaxman S, Fowkes FGR, Freedman G, Freeman MK, Gakidou E, 702 Ghosh S, Giovannucci E, Gmel G, Graham K, Grainger R, Grant B, Gunnell D, 703 Gutierrez HR, Hall W, Hoek HW, Hogan A, Hosgood HD, Hoy D, Hu H, Hubbell BJ, 704 Hutchings SJ, Ibeanusi SE, Jacklyn GL, Jasrasaria R, Jonas JB, Kan H, Kanis JA, 705 Kassebaum N, Kawakami N, Khang Y-H, Khatibzadeh S, Khoo J-P, Kok C, Laden F, 706 Lalloo R, Lan Q, Lathlean T, Leasher JL, Leigh J, Li Y, Lin JK, Lipshultz SE, London S, 707 Lozano R, Lu Y, Mak J, Malekzadeh R, Mallinger L, Marcenes W, March L, Marks R, 708 Martin R, McGale P, McGrath J, Mehta S, Mensah GA, Merriman TR, Micha R, Michaud 709 C, Mishra V, Mohd Hanafiah K, Mokdad AA, Morawska L, Mozaffarian D, Murphy T, 710 Naghavi M, Neal B, Nelson PK, Nolla JM, Norman R, Olives C, Omer SB, Orchard J, 711 Osborne R, Ostro B, Page A, Pandey KD, Parry CDH, Passmore E, Patra J, Pearce N, 712 Pelizzari PM, Petzold M, Phillips MR, Pope D, Pope CA, Powles J, Rao M, Razavi H, 713 Rehfuess EA, Rehm JT, Ritz B, Rivara FP, Roberts T, Robinson C, Rodriguez-Portales 714 JA, Romieu I, Room R, Rosenfeld LC, Roy A, Rushton L, Salomon JA, Sampson U, 715 Sanchez-Riera L, Sanman E, Sapkota A, Seedat S, Shi P, Shield K, Shivakoti R, Singh 716 GM, Sleet DA, Smith E, Smith KR, Stapelberg NJC, Steenland K, Stöckl H, Stovner LJ, 717 Straif K, Straney L, Thurston GD, Tran JH, Van Dingenen R, van Donkelaar A, Veerman 718 JL, Vijayakumar L, Weintraub R, Weissman MM, White RA, Whiteford H, Wiersma ST, 719 Wilkinson JD, Williams HC, Williams W, Wilson N, Woolf AD, Yip P, Zielinski JM, Lopez 720 AD, Murray CJL, Ezzati M, AlMazroa MA, Memish ZA. A comparative risk assessment 721 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 31 of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 722 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 723 2010. Lancet (London, England). 2012;380(9859):2224-60. doi: 10.1016/S0140-724 6736(12)61766-8. PubMed PMID: 23245609; PMCID: PMC4156511. 725 27. Whelton PK, Carey RM, Aronow WS, Casey DE, Jr., Collins KJ, Dennison 726 Himmelfarb C, DePalma SM, Gidding S, Jamerson KA, Jones DW, MacLaughlin EJ, 727 Muntner P, Ovbiagele B, Smith SC, Jr., Spencer CC, Stafford RS, Taler SJ, Thomas RJ, 728 Williams KA, Sr., Williamson JD, Wright JT, Jr. 2017 729 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the 730 Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A 731 Report of the American College of Cardiology/American Heart Association Task Force 732 on Clinical Practice Guidelines. Hypertension. 2018;71(6):e13-e115. Epub 20171113. 733 doi: 10.1161/HYP.0000000000000065. PubMed PMID: 29133356. 734 28. Xu J, Murphy SL, Kochanek KD, Arias E. Mortality in the United States, 2021. 735 NCHS Data Brief. 2022(456):1-8. PubMed PMID: 36598387. 736 29. Burnier M, Damianaki A. Hypertension as Cardiovascular Risk Factor in Chronic 737 Kidney Disease. Circ Res. 2023;132(8):1050-63. Epub 20230413. doi: 738 10.1161/CIRCRESAHA.122.321762. PubMed PMID: 37053276. 739 30. Pacholko A, Iadecola C. Hypertension, Neurodegeneration, and Cognitive 740 Decline. Hypertension. 2024;81(5):991-1007. Epub 20240301. doi: 741 10.1161/HYPERTENSIONAHA.123.21356. PubMed PMID: 38426329; PMCID: 742 PMC11023809. 743 31. Sariban-Sohraby S, Benos DJ. The amiloride-sensitive sodium channel. Am J 744 Physiol. 1986;250(2 Pt 1):C175-90. doi: 10.1152/ajpcell.1986.250.2.C175. PubMed 745 PMID: 2420186. 746 32. Frindt G, Meyerson JR, Satty A, Scandura JM, Palmer LG. Expression of ENaC 747 subunits in epithelia. J Gen Physiol. 2022;154(10). Epub 20220808. doi: 748 10.1085/jgp.202213124. PubMed PMID: 35939271; PMCID: PMC9387651. 749 33. Shehata MF. The Epithelial Sodium Channel alpha subunit (alpha ENaC) 750 alternatively spliced form "b" in Dahl rats: What's next? Int Arch Med. 2010;3:14. Epub 751 20100706. doi: 10.1186/1755-7682-3-14. PubMed PMID: 20604958; PMCID: 752 PMC2909934. 753 34. Krueger B, Schlotzer-Schrehardt U, Haerteis S, Zenkel M, Chankiewitz VE, 754 Amann KU, Kruse FE, Korbmacher C. Four subunits (alphabetagammadelta) of the 755 epithelial sodium channel (ENaC) are expressed in the human eye in various locations. 756 Invest Ophthalmol Vis Sci. 2012;53(2):596-604. Epub 20120202. doi: 10.1167/iovs.11-757 8581. PubMed PMID: 22167092. 758 35. Noreng S, Bharadwaj A, Posert R, Yoshioka C, Baconguis I. Structure of the 759 human epithelial sodium channel by cryo-electron microscopy. Elife. 2018;7. Epub 760 2018/09/27. doi: 10.7554/eLife.39340. PubMed PMID: 30251954; PMCID: 761 PMC6197857. 762 36. Noreng S, Posert R, Bharadwaj A, Houser A, Baconguis I. Molecular principles of 763 assembly, activation, and inhibition in epithelial sodium channel. Elife. 2020;9. Epub 764 20200730. doi: 10.7554/eLife.59038. PubMed PMID: 32729833; PMCID: PMC7413742. 765 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 32 37. Houser A, Baconguis I. Structural insights into subunit-dependent functional 766 regulation in epithelial sodium channels. Structure. 2024. Epub 20241205. doi: 767 10.1016/j.str.2024.11.013. PubMed PMID: 39667931. 768 38. Fyfe GK, Canessa CM. Subunit composition determines the single channel 769 kinetics of the epithelial sodium channel. J Gen Physiol. 1998;112(4):423-32. Epub 770 1998/10/06. PubMed PMID: 9758861; PMCID: PMC2229421. 771 39. Kleyman TR, Carattino MD, Hughey RP. ENaC at the cutting edge: regulation of 772 epithelial sodium channels by proteases. J Biol Chem. 2009;284(31):20447-51. Epub 773 2009/04/30. doi: 10.1074/jbc.R800083200. PubMed PMID: 19401469; PMCID: 774 PMC2742807. 775 40. Kellenberger S, Schild L. Epithelial sodium channel/degenerin family of ion 776 channels: a variety of functions for a shared structure. Physiological Reviews. 777 2002;82(3):735-67. doi: 10.1152/physrev.00007.2002. PubMed PMID: 12087134. 778 41. Rossier BC, Stutts MJ. Activation of the Epithelial Sodium Channel (ENaC) by 779 Serine Proteases. Annual Review of Physiology. 2009;71(1):361-79. doi: 780 10.1146/annurev.physiol.010908.163108. PubMed PMID: 18928407. 781 42. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, Boucher 782 RC. CFTR as a cAMP-dependent regulator of sodium channels. Science. 783 1995;269(5225):847-50. doi: 10.1126/science.7543698. PubMed PMID: 7543698. 784 43. Staub O, Abriel H, Plant P, Ishikawa T, Kanelis V, Saleki R, Horisberger JD, 785 Schild L, Rotin D. Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. 786 Kidney Int. 2000;57(3):809-15. doi: 10.1046/j.1523-1755.2000.00919.x. PubMed PMID: 787 10720933. 788 44. Lang F, Shumilina E. Regulation of ion channels by the serum- and 789 glucocorticoid-inducible kinase SGK1. The FASEB Journal. 2013;27(1):3-12. doi: 790 10.1096/fj.12-218230. 791 45. Hill WG, An B, Johnson JP. Endogenously expressed epithelial sodium channel 792 is present in lipid rafts in A6 cells. J Biol Chem. 2002;277(37):33541-4. Epub 20020806. 793 doi: 10.1074/jbc.C200309200. PubMed PMID: 12167633. 794 46. Hill WG, Butterworth MB, Wang H, Edinger RS, Lebowitz J, Peters KW, Frizzell 795 RA, Johnson JP. The epithelial sodium channel (ENaC) traffics to apical membrane in 796 lipid rafts in mouse cortical collecting duct cells. J Biol Chem. 2007;282(52):37402-11. 797 Epub 20071010. doi: 10.1074/jbc.M704084200. PubMed PMID: 17932048. 798 47. Rossier BC. Hormonal regulation of the epithelial sodium channel ENaC: N or 799 P(o)? J Gen Physiol. 2002;120(1):67-70. doi: 10.1085/jgp.20028638. PubMed PMID: 800 12084776; PMCID: PMC2311401. 801 48. Kawate T, Gouaux E. Fluorescence-detection size-exclusion chromatography for 802 precrystallization screening of integral membrane proteins. Structure. 2006;14(4):673-803 81. doi: 10.1016/j.str.2006.01.013. PubMed PMID: 16615909. 804 49. Jain A, Liu R, Xiang YK, Ha T. Single-molecule pull-down for studying protein 805 interactions. Nat Protoc. 2012;7(3):445-52. Epub 20120209. doi: 806 10.1038/nprot.2011.452. PubMed PMID: 22322217; PMCID: PMC3654178. 807 50. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A. A variant of 808 yellow fluorescent protein with fast and efficient maturation for cell-biological 809 applications. Nat Biotechnol. 2002;20(1):87-90. doi: 10.1038/nbt0102-87. PubMed 810 PMID: 11753368. 811 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 33 51. Cordingley MG, Callahan PL, Sardana VV, Garsky VM, Colonno RJ. Substrate 812 requirements of human rhinovirus 3C protease for peptide cleavage in vitro. J Biol 813 Chem. 1990;265(16):9062-5. PubMed PMID: 2160953. 814 52. Inc. N. Accelerate your research with the latest in computational biology 815 Wilmington, Delaware2022. Available from: https://neurosnap.ai/. 816 53. Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X. 817 Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 818 2013;23(5):720-3. Epub 20130402. doi: 10.1038/cr.2013.46. PubMed PMID: 23545779; 819 PMCID: PMC3641603. 820 54. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. 821 One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-822 mediated genome engineering. Cell. 2013;153(4):910-8. Epub 20130502. doi: 823 10.1016/j.cell.2013.04.025. PubMed PMID: 23643243; PMCID: PMC3969854. 824 55. Barker PM, Nguyen MS, Gatzy JT, Grubb B, Norman H, Hummler E, Rossier B, 825 Boucher RC, Koller B. Role of gammaENaC subunit in lung liquid clearance and 826 electrolyte balance in newborn mice. Insights into perinatal adaptation and 827 pseudohypoaldosteronism. J Clin Invest. 1998;102(8):1634-40. doi: 10.1172/JCI3971. 828 PubMed PMID: 9788978; PMCID: PMC509015. 829 56. Picard N, Eladari D, El Moghrabi S, Planes C, Bourgeois S, Houillier P, Wang Q, 830 Burnier M, Deschenes G, Knepper MA, Meneton P, Chambrey R. Defective ENaC 831 processing and function in tissue kallikrein-deficient mice. J Biol Chem. 832 2008;283(8):4602-11. Epub 20071217. doi: 10.1074/jbc.M705664200. PubMed PMID: 833 18086683. 834 57. Kleyman TR, Cragoe EJ, Jr. Amiloride and its analogs as tools in the study of ion 835 transport. J Membr Biol. 1988;105(1):1-21. Epub 1988/10/01. PubMed PMID: 2852254. 836 58. Hattori M, Hibbs RE, Gouaux E. A fluorescence-detection size-exclusion 837 chromatography-based thermostability assay for membrane protein precrystallization 838 screening. Structure. 2012;20(8):1293-9. doi: 10.1016/j.str.2012.06.009. PubMed PMID: 839 22884106; PMCID: PMC3441139. 840 59. Axelrod D. Chapter 7: Total internal reflection fluorescence microscopy. Methods 841 Cell Biol. 2008;89:169-221. doi: 10.1016/S0091-679X(08)00607-9. PubMed PMID: 842 19118676. 843 60. Clark S, Elferich J, Gai J, Goehring A, Mitra J, Ha T, Gouaux E. Strategy for 844 Compositional Analysis of the Hair Cell Mechanotransduction Complex Using TIRF 845 Microscopy. Microsc Microanal. 2019;25(Suppl 2):1266-7. Epub 20190805. doi: 846 10.1017/s1431927619007062. PubMed PMID: 32025193; PMCID: PMC7001662. 847 61. Kubala MH, Kovtun O, Alexandrov K, Collins BM. Structural and thermodynamic 848 analysis of the GFP:GFP-nanobody complex. Protein Sci. 2010;19(12):2389-401. doi: 849 10.1002/pro.519. PubMed PMID: 20945358; PMCID: PMC3009406. 850 62. Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, 851 Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TW, Jr. mScarlet: a bright 852 monomeric red fluorescent protein for cellular imaging. Nat Methods. 2017;14(1):53-6. 853 Epub 20161121. doi: 10.1038/nmeth.4074. PubMed PMID: 27869816. 854 63. Hammond C, Braakman I, Helenius A. Role of N-linked oligosaccharide 855 recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. 856 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 34 Proc Natl Acad Sci U S A. 1994;91(3):913-7. doi: 10.1073/pnas.91.3.913. PubMed 857 PMID: 8302866; PMCID: PMC521423. 858 64. Vassilakos A, Cohen-Doyle MF, Peterson PA, Jackson MR, Williams DB. The 859 molecular chaperone calnexin facilitates folding and assembly of class I 860 histocompatibility molecules. EMBO J. 1996;15(7):1495-506. PubMed PMID: 8612572; 861 PMCID: PMC450057. 862 65. Manganas LN, Trimmer JS. Calnexin regulates mammalian Kv1 channel 863 trafficking. Biochem Biophys Res Commun. 2004;322(2):577-84. doi: 864 10.1016/j.bbrc.2004.06.182. PubMed PMID: 15325269. 865 66. Garty H, Palmer LG. Epithelial sodium channels: function, structure, and 866 regulation. Physiological Reviews. 1997. PubMed PMID: 9CD90C0A-7172-42BE-8B76-867 030D356A3C0B. 868 67. Jeong H, Clark S, Goehring A, Dehghani-Ghahnaviyeh S, Rasouli A, Tajkhorshid 869 E, Gouaux E. Structures of the TMC-1 complex illuminate mechanosensory 870 transduction. Nature. 2022;610(7933):796-803. Epub 20221012. doi: 10.1038/s41586-871 022-05314-8. PubMed PMID: 36224384; PMCID: PMC9605866. 872 68. Goehring A, Lee C-H, Wang KH, Michel JC, Claxton DP, Baconguis I, Althoff T, 873 Fischer S, Garcia KC, Gouaux E. Screening and large-scale expression of membrane 874 proteins in mammalian cells for structural studies. Nature Protocols. 2014;9(11):2574-875 85. doi: 10.1038/nprot.2014.173. PubMed PMID: 25299155; PMCID: PMC4291175. 876 69. Sorensen MV, Grossmann S, Roesinger M, Gresko N, Todkar AP, Barmettler G, 877 Ziegler U, Odermatt A, Loffing-Cueni D, Loffing J. Rapid dephosphorylation of the renal 878 sodium chloride cotransporter in response to oral potassium intake in mice. Kidney Int. 879 2013;83(5):811-24. Epub 20130227. doi: 10.1038/ki.2013.14. PubMed PMID: 880 23447069. 881 70. Grimm PR, Taneja TK, Liu J, Coleman R, Chen YY, Delpire E, Wade JB, Welling 882 PA. SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-883 specific manner. J Biol Chem. 2012;287(45):37673-90. Epub 20120912. doi: 884 10.1074/jbc.M112.402800. PubMed PMID: 22977235; PMCID: PMC3488044. 885 886 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 35 Figure legends 887 Figure 1. Immunofluorescence staining of kidney tissue from homozygous 888 ENaCγ-VF mice. 889 a. Schematic of the tagged ENaCγ subunit produced from the ENaCγ-VF allele, which 890 includes a C-terminal mVenus, 3C protease site, and 3xFLAG tag. b. Representative 891 immunofluorescence image showing co-localization of mVenus and ENaCγ in kidney 892 tissue. mVenus signal was detected using a FITC-conjugated anti-GFP primary 893 antibody (goat, Rockland, 1:100) and Alexa Fluor 488 donkey anti-goat secondary 894 antibody. Endogenous ENaCγ was detected using a rabbit anti-ENaCγ primary antibody 895 (StressMarq, 1:500) targeting the C-terminus, and Alexa Fluor 647 donkey anti-rabbit 896 secondary antibody. DAPI is 4’,6-diamidino-2-phenylindole. c. Close-up view 897 highlighting localization of tagged ENaCγ. 898 Figure 2. The ENaCγ-VF mouse line is physiologically indistinguishable from 899 wild-type at baseline. 900 a. Table showing plasma sodium (Na+) and potassium (K⁺) concentrations in wild-type 901 C57BL/6 and homozygous ENaCγ-VF mice under baseline conditions. b. Amiloride 902 response assay in wild-type C57BL/6 and ENaCγ-VF mice. The graph shows individual 903 paired responses to vehicle and amiloride for each mouse, with overlaid bars indicating 904 mean ± SEM (n = 6 per group). A two-way ANOVA revealed a significant effect of 905 amiloride treatment compared with vehicle (***p < 0.0001), but no significant difference 906 between genotypes (p = 0.4533), indicating that the tagged allele supports normal 907 ENaC function. c. Saturation binding of [³H]benzamil to kidney membranes. Increasing 908 concentrations of [³H]benzamil (0.5-300 nM) were incubated with kidney membranes for 909 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 36 2 hours min at 22 °C. Specific binding (circles) was determined by subtracting 910 nonspecific binding measured in the presence of 100 µM unlabeled phenamil. Data 911 represent one experiment performed in triplicate; points show mean ± SD of triplicate 912 measurements. The curve was fit with a one-site specific binding model using nonlinear 913 regression (GraphPad Prism) to obtain Kd. 914 Figure 3. The ENaCγ-VF mouse enables subunit-specific detection of native ENaC 915 complexes using FSEC and SiMPull. 916 a. Schematic workflow illustrating the experimental pipeline. Tissues (lung, kidney, 917 colon) were harvested from ENaCγ-VF mice, homogenized in TRIS-based buffer, and 918 followed by solubilization. Cleared supernatants were injected onto a Superose 6 919 Increase column for FSEC analysis, and fractions corresponding to high-molecular-920 weight complexes were subsequently analyzed by SiMPull. b. Representative FSEC 921 traces showing mVenus fluorescence from lung, kidney, and colon lysates. Fluorescent 922 peaks indicate the presence of γ-containing ENaC complexes in each tissue. c. 923 Representative TIRF images of SiMPull experiments from lung lysates. mVenus-tagged 924 ENaC complexes were captured using a biotinylated anti-GFP/mVenus nanobody. A 925

control image was collected from a flow chamber lacking the nanobody to 926 demonstrate specificity. d. Quantification of SiMPull signal from lung, kidney, and colon 927 tissues. Each condition represents data pooled from 40 TIRF images per sample, 928 analyzed using the ComDet plugin in ImageJ. Statistical analysis was performed in 929 GraphPad Prism. 930 Figure 4. Dual-subunit detection of native ENaC complexes using 7B1-mScarlet 931 and the ENaCγ-VF mouse line. 932 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 37 a. Cryo-EM map of human ENaC in complex with two Fabs, 10D4 and 7B1, shown in 933 side and top-down views. In the top-down view, a red box highlights the interaction site 934 between 7B1 and the α subunit. b. Sequence alignment of the experimentally 935 determined 7B1 epitope region in human and mouse ENaCα subunits, revealing high 936 conservation and supporting the potential for cross-reactivity with mouse ENaC. c. 937 Schematic representation of the 7B1-mScarlet Fab construct used to detect the ENaC α 938 subunit. The variable regions of the 7B1 heavy and light chains were expressed as a 939 Fab fragment in HEK293 cells, with mScarlet fused to the C-terminus of the heavy 940 chain. d. FSEC trace monitoring mVenus fluorescence from lung lysates of ENaCγ-VF 941 mice, showing the elution profile of γ-containing ENaC complexes. Dotted lines indicate 942 the elution range for mVenus-tagged complexes (11-16 mL). e. FSEC trace from the 943 same sample following 7B1-mScarlet fluorescence, revealing the elution profile of α-944 containing ENaC complexes. Dashed lines show that the α-associated signal is 945 restricted to a narrower elution range (11-13 mL), suggesting that α-γ co-assemblies 946 represent a more defined subset of the total γ-containing complexes. f. Overlay of the 947 FSEC traces shown in panels d and e, plotted on the same axes to facilitate direct 948 comparison of elution profiles and peak positions. The inset shows the full normalized 949 traces for each condition. The main panel displays a magnified view of the gray-shaded 950 region indicated in the inset, highlighting the relative alignment of the major peaks. 951 Figure 5. ENaC complex purification enables tissue-specific interactome analysis 952 by mass spectrometry. 953 a. Schematic workflow illustrating the purification pipeline. Tissues (lung, kidney, colon) 954 from ENaCγ-VF mice were solubilized under non-denaturing conditions. γ-containing 955 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 38 ENaC complexes were enriched by affinity capture via the C-terminal FLAG tag, eluted 956 by 3C protease cleavage, and analyzed by mass spectrometry. b. Representative FSEC 957 traces from lung, kidney, and colon showing mVenus fluorescence, confirming 958 successful enrichment of γ-containing complexes. The gray shaded region indicates the 959 fractions that were collected, pooled, and used for mass spectrometry analysis. A 960 second prominent peak is observed and corresponds to background fluorescence 961 detected in kidney tissue, independent of ENaC-containing complexes. c. Mass 962 spectrometry results identifying proteins that co-purify with ENaCγ from each tissue. 963 Data reveal both shared and tissue-enriched interactors, highlighting the power of this 964 approach to define native ENaC-associated complexes in a tissue-specific context. 965 Figure S1. Immunofluorescence staining of kidney tissue from heterozygous 966 ENaCγ-VF mice. 967 a. Representative immunofluorescence image showing co-localization of mVenus, 968 ENaCγ, and aquaporin-2 in kidney tissue. mVenus signal was detected using a FITC-969 conjugated anti-GFP primary antibody (goat, Rockland, 1:100) and Alexa Fluor 488 970 donkey anti-goat secondary antibody. Endogenous ENaCγ was detected using a rabbit 971 anti-ENaCγ primary antibody (StressMarq, 1:500) targeting the C-terminus, and Alexa 972 Fluor 647 donkey anti-rabbit secondary antibody. Aquaporin-2 (AQP2) was stained 973 using a mouse anti-AQP2 primary antibody (Santa Cruz, 1:50) and Alexa Fluor 555 974 donkey anti-mouse secondary antibody. b. Representative immunofluorescence image 975 showing co-localization of mVenus, ENaCα, and pNCC in kidney tissue. mVenus signal 976 was detected using a FITC-conjugated anti-GFP primary antibody (goat, Rockland, 977 1:100) and Alexa Fluor 488 donkey anti-goat secondary antibody. Endogenous ENaCα 978 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 39 was detected using a rabbit anti-ENaCα primary antibody (generous gift of J. Loffing69) 979 targeting the C-terminus, and Alexa Fluor 647 donkey anti-rabbit secondary antibody. 980 The phosphorylated form of the Na⁺-Cl⁻ cotransporter (NCC) was stained using a 981 chicken anti-NCC (generously provided by J. Wade70) and Cy3 donkey anti-chicken 982 secondary antibody. c. Close-up view highlighting localization of tagged mVenus, 983 ENaCα, and pNCC. * indicate DCT1 (pNCC+/ENaCα-); + indicate DCT2 984 (pNCC+/ENaCα+). 985 Figure S2. Validation of 7B1 -mScarlet as a tool for detecting mouse ENaC α. 986 a. Schematic of the recombinant mouse ENaC subunit constructs used for validation. The 987 α and β subunits were expressed with wild -type sequences, while the γ subunit was C-988 terminally fused to mVenus, mirroring the configuration in ENaC γ-VF mice. b, c. FSEC 989 traces showing mVenus (b) and mScarlet fluorescence (c) from lysates of cells expressing 990 recombinant mouse ENaC. Co -elution of the two signals confirms that 7B1 -mScarlet 991 binds specifically to complexes containing ENaC α, validating its use in detecting α 992 subunits within γ-containing assemblies. d. Schematic illustration of ENaC complexes 993 located inside the cell and in the plasma membrane. Only surface ENaC is exposed to 994 membrane-impermeable biotin. e. FSEC traces of purified surface -expressed ENaC 995 protein (black trace) and intracellular ENaC pool (blue trace) monitored on the mScarlet 996 channel fused to the 7B1 Fab. The difference in peak height is due to preexisting 997 differences in relative abundance and not a difference in 7B1 recognition. 998 Figure S3. Control purification workflow using wild-type mice to assess non-999 specific binding. 1000 Schematic of the purification workflow applied to wild-type mouse tissues, performed in 1001 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint 40 parallel with the ENaCγ-VF workflow. Although the protocol is identical, including tissue 1002 solubilization, anti-FLAG affinity capture, and protease elution, wild-type mice lack the 1003 C-terminal 3xFLAG tag on ENaCγ and therefore cannot be captured by the anti-FLAG 1004 resin. This control serves to identify proteins that may non-specifically bind to the affinity 1005 resin and distinguishes true ENaC-associated interactors from background 1006 contaminants in the ENaCγ-VF purification experiments. 1007 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint Figure 1 a ENaC γ mVenus 3C protease site 3xFLAG ENaCγ-VF b mV enus ENaCγ DAPI Merged c mV enus ENaCγ DAPI Merged 500 μm 50 μm .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint Figure 2 a ENaCγ-VF (N=3) mmol/L 147 (2.65) 3.80 (0.520) WT (N=3) mmol/L 146 (1.73) 3.73 (0.351) Na+ Mean (SD) K+ Mean (SD) b c Urine Na+:K+ ratio (mmol/mmol) 0 5 10 15 WT ENaCγ-VF Vehicle Amiloride*** *** NS 0 100 200 300 0 500 1000 1500 2000 nM Radioligand Specific binding (cpm) ENaCγ-VF WT .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint Figure 3 PEG Glass coverslip Biotin mVenus Streptavidin Biotinylated GFP nanobody ENaCγ a ENaCγ-VF SEC column Fluorometer Elution volume (mL) Protein complex size Large Small Void Fluorescence (a.u.) mVenus fluorescence b BackgroundmVenus c lungs kidneys colon d mVenus

mVenus

mVenus

0 500 1000 1500Number of molecules **** **** ****p<0.0001 **** 12.5 15 Elution Volume (ml) ColonColon KidneysKidneys LungsLungs 3xFLAG .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint Figure 4 7B1 light chain 7B1 heavy chain mScarlet c d 0 10 20 30 Elution Volume (mL) mVenus mVenus fluorescence mScarlet fluorescence Normalized mVenus e f 0 10 20 30 Elution Volume (mL) mScarlet mScarletExcess 7B1- mScarlet a Out γ β α γ β α 7B1 Fab 10D4 Fab 10D4 Fab 7B1 Fab In 90° b hENaCα LPETLPSLE mENaCα LPDTSPALE 263 271 290 298 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint Figure 5 10 15 20 ENaCγ-VF lungs ENaCγ-VF tissue Supernatant Affinity isolation FSEC purification Mass spectrometry Solubilize tissue Anti-FLAG resin 3xFLAG 3C protease cleavage Concentrate eluted fractions mVenus fluorescence Elution volume (mL) 10 15 20 ENaCγ-VF kidneys mVenus fluorescence Elution volume (mL) 10 15 20 ENaCγ-VF colon mVenus fluorescence Elution volume (mL) ba c WT ENaCγ-VF 0 0 0 0 0 0 8 3 3 4 2 0 KidneyKidney Lungs Colon SCNNG_MOUSE 33 SCNNB_MOUSE 25 SCNNA_MOUSE 14 DHI2_MOUSE 19 CALX_MOUSE 14 NED4L_MOUSE 11 112 136 52 46 0 14 Pool fractions .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint