{"paper_id":"2ace80cf-6a63-4b50-a1ea-cfa3a6105c00","body_text":"1Vollum Institute, Oregon Health & Science University, Portland, OR 97239 \n2Division of Nephrology and Hypertension, Department of Medicine, Oregon Health and \nScience University, Portland, Oregon, 97239 \n3Department of Chemical Physiology and Biochemistry, Oregon Health and Science \nUniversity, Portland, Oregon, 97239 \n4Correspondence: Isabelle Baconguis, bacongui@ohsu.edu \n \nDifferential Assembly of Native ENaC Complexes Across Mouse Epithelial Tissues \n \n \n \n \nArpita Bharadwaj1, Joshua Curry2, Xiao-Tong Su2, Romina Barria Maturana1, James A. \nMcCormick2,3, David H. Ellison2,3, and Isabelle Baconguis1,4 \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 2 \nAbstract 1 \nThe epithelial sodium channel (ENaC) governs sodium and fluid absorption in the lung, 2 \nkidney, and colon, but the organization of native ENaC complexes has remained 3 \ndifficult to define because of their low abundance and biochemical instability. To enable 4 \ndirect analysis of native assemblies, we generated a knock -in mouse in which the 5 \nendogenous γ subunit is fused at its C terminus to mVenus, a 3C protease cleavage 6 \nsite, and a 3xFLAG epitope (ENaCγ-VF). The tag preserves physiological ENaC 7 \nfunction, as ENaCγ-VF mice display normal electrolyte handling, benzamil affinity, and 8 \namiloride-sensitive Na⁺:K⁺ responses indistinguishable from wild -type animals. Using 9 \nfluorescence-detection size-exclusion chromatography and single -molecule pull-down, 10 \nwe directly monitor intact native ENaC complexes from lung, kidney, and colon and 11 \nuncover marked tissue-to-tissue differences in channel abundance and apparent 12 \ncomplex size. Dual-color analysis with a fluorescent Fab against ENaCα marks fully 13 \nassembled αβγ channels, while γ-based fluorescence reports the broader population of 14 \nγ-containing assemblies. In combination, the ENaCγ -VF line provides a biochemical 15 \nanchor for identifying regulatory and trafficking proteins that co -purify with native ENaC 16 \ncomplexes. These data show that ENaC architecture in vivo is heterogeneous, and 17 \nestablish ENaCγ-VF mice as a platform for dissecting how epithelial environments 18 \nshape ENaC assembly, composition, and regulation.  19 \n 20 \n 21 \n 22 \n 23 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 3 \nIntroduction 24 \nCommunication across epithelial barriers depends critically on the controlled movement 25 \nof sodium ions, a process in which the epithelial sodium channel (ENaC) plays a central 26 \nand indispensable role. Situated at the apical membrane of epithelial cells, ENaC drives 27 \nsodium entry that underlies extracellular fluid balance, blood pressure regulation, and 28 \nairway surface hydration1-4. Its activity is required for efficient alveolar fluid clearance in 29 \nthe lung5, 6, sodium reabsorption in the distal nephron1, and electrolyte absorption in the 30 \ncolon7. Dysregulation of ENaC function contributes to diseases such as cystic fibrosis8 31 \nand pseudohypoaldosteronism9-12, as well as multifactorial disorders including 32 \npulmonary edema13 and hypertension14-25. Hypertension alone affects more than one 33 \nbillion individuals worldwide and remains the leading modifiable risk factor for 34 \ncardiovascular disease26-28, chronic kidney disease29, and cognitive decline30. These 35 \nclinical associations have long underscored the need to understand how ENaC is 36 \nassembled, regulated, and stabilized within its native environment. 37 \n 38 \nBefore recombinant expression was feasible, early conceptual models of ENaC biology 39 \nemerged largely from electrophysiological studies of transepithelial transport, which 40 \ninferred the presence of a selective sodium channel from its amiloride sensitivity and 41 \nionic selectivity31. These foundational observations established the physiological role of 42 \nENaC long before its molecular identity was known. The subsequent cloning of the α, β, 43 \nand γ subunits provided the first molecular description for the channel and revealed a 44 \nfamily of homologous proteins with conserved topology across vertebrate epithelia32-34. 45 \nRecombinant expression systems soon enabled functional dissection through 46 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 4 \nmutagenesis, proteolytic activation studies, and single-channel recordings, offering 47 \nhigh-resolution insights into ENaC architecture35-37, gating38, and inhibition39. 48 \n 49 \nYet these advances captured only a portion of ENaC biology. Within native tissues, 50 \nENaC expression is highly compartmentalized40, its maturation requires sequential 51 \ncleavage by distinct proteases39, 41, and its surface abundance is dynamically regulated 52 \nby a network of accessory proteins and ubiquitin ligases1, 42-44. Electrophysiological and 53 \ntranscriptomic analyses have yielded valuable perspectives, but neither directly resolves 54 \nhow ENaC subunits assemble into functional complexes, nor how regulatory factors 55 \nshape the architecture and stability of those assemblies. As a result, the molecular 56 \norganization of ENaC in situ, its subunit stoichiometry, tissue-specific variability, and 57 \nregulatory context, remain only partially defined. A fundamental obstacle has been the 58 \nabsence of tools enabling direct detection, purification, and visualization of ENaC under 59 \nnon-denaturing conditions. ENaC is expressed at exceptionally low abundance, 60 \nbiochemically labile, and embedded within lipid environments that complicate 61 \nsolubilization45-47. Attempts to recover native ENaC have traditionally yielded insufficient 62 \nquantities for systematic analysis, limiting the field to indirect methods or recombinant 63 \nmodels that cannot fully replicate the physiological assembly landscape. 64 \n 65 \nTo address this longstanding bottleneck, we developed a genetically engineered knock-66 \nin mouse line (ENaCγ-VF) in which the endogenous γ subunit is fused at its C terminus 67 \nto mVenus, followed by a 3C protease site and a 3xFLAG epitope. This multifunctional 68 \ntag enables fluorescence-based detection, affinity purification, and gentle protease-69 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 5 \nmediated elution of intact ENaC complexes while preserving native expression levels, 70 \nregulatory control, and tissue specificity. Here, we validate that the tagged γ subunit 71 \nsupports normal ENaC function in vivo and demonstrate that the ENaCγ-VF line is 72 \ncompatible with fluorescence-detection size-exclusion chromatography (FSEC)48 and 73 \nsingle-molecule pull-down (SiMPull)49, permitting direct quantification of native ENaC 74 \nassemblies isolated from lung, kidney, and colon. In parallel, we developed a 75 \nfluorescent Fab fragment targeting ENaCα, enabling dual-color, subunit-resolved 76 \nanalysis of ENaC architecture in native lysates. These tools establish a robust platform 77 \nfor dissecting ENaC assembly, composition, and regulation directly in physiological 78 \ncontexts. Because ENaC activity must be tuned to the distinct transport demands of 79 \ndifferent epithelia, tissue-specific differences in subunit assembly and associations with 80 \nregulatory partners are likely to shape Na+ transport capacity and hormonal 81 \nresponsiveness1-4, 39, 41-44.  82 \n 83 \nResults 84 \n 85 \nGeneration of the ENaCγ-VF mouse line 86 \nTo enable detection and purification of native ENaC complexes, we designed a knock-in 87 \nmouse line in which the endogenous Scnn1g locus (encoding ENaCγ) was modified to 88 \nincorporate a C-terminal mVenusQ69M fluorophore, a 3C protease recognition site, and a 89 \n3xFLAG epitope50-52 (Fig. 1a). This multifunctional design enables direct fluorescence 90 \ndetection, native-state affinity isolation, and protease-mediated elution of intact ENaC 91 \nassemblies. The ENaCγ-VF allele was generated at the Jackson Laboratory using 92 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 6 \nCRISPR/Cas9 to insert the tag in-frame at the endogenous Scnn1g stop codon53, 54. 93 \nInitial characterization was performed in heterozygous ENaCγ-VF+/- mice to assess 94 \nwhether C-terminal tagging of γENaC perturbed channel expression, trafficking, or 95 \nsubcellular localization in the presence of an unmodified endogenous allele. 96 \nHeterozygous ENaCγ-VF+/- mice exhibited normal appearance and growth, and 97 \nimmunostaining of kidney sections confirmed correct apical localization of the tagged γ 98 \nsubunit within the distal nephron (Fig. S1), consistent with preserved trafficking and 99 \nexpression40. 100 \n 101 \nA central requirement for using this model to study native ENaC is that the modified γ 102 \nsubunit must independently support physiological channel function on its own. Because 103 \nScnn1g knockout is perinatally lethal55, viability in the homozygous state provides a 104 \nstringent functional test of the tagged allele. Homozygous ENaCγ-VF/ENaCγ-VF mice 105 \nwere viable into adulthood, indicating that the mVenus-3C-3xFLAG fusion does not 106 \ndisrupt essential ENaC activity. These animals therefore served as the foundation for all 107 \nsubsequent physiological, biochemical, and biophysical analyses. 108 \n 109 \nValidation of the ENaCγ-VF mouse line 110 \nWe validated the expression and localization of the tagged γ subunit in kidney tissue, 111 \nwhere ENaC plays a well-defined role in sodium reabsorption. Immunostaining showed 112 \nthat mVenus-tagged γ was appropriately restricted to the distal nephron, consistent with 113 \nthe known distribution of native ENaC40 (Fig. 1b, c). Blood electrolyte levels in ENaCγ-114 \nVF mice were within the normal physiological range (Fig. 2a). To assess whether the 115 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 7 \ntagged γ subunit supports normal channel function in vivo, we examined the response 116 \nof ENaCγ-VF mice to amiloride, a well-characterized ENaC inhibitor56. As expected, 117 \npharmacological blockade increased the urinary Na⁺:K⁺ ratio, reflecting inhibition of 118 \nENaC-mediated sodium reabsorption and potassium secretion in the distal nephron. 119 \nENaCγ-VF mice exhibited an amiloride-induced increase in Na⁺:K⁺ ratio 120 \nindistinguishable from wild-type controls (Fig. 2b), indicating preserved ENaC function. 121 \nThese findings demonstrate that homozygous ENaCγ-VF mice maintain normal 122 \nelectrolyte handling and are phenotypically comparable to wild-type littermates under 123 \nbasal conditions. 124 \n 125 \nTo complement these physiological measurements with a direct biochemical 126 \nassessment of channel pharmacology, we performed radioligand filter-binding assays 127 \nusing tritiated [³H]-benzamil, a high-affinity ENaC antagonist57. Kidney membranes were 128 \nselected for these experiments because renal ENaC activity underlies the in vivo 129 \namiloride challenge and provides a robust, well-defined tissue context in which ENaC-130 \ndependent sodium transport can be directly interrogated. Kidney membrane fractions 131 \nwere isolated, incubated with increasing concentrations of [³H]-benzamil, and filtered to 132 \nseparate receptor-bound from free radioligand. Specific benzamil binding was readily 133 \ndetectable in both wild-type and ENaCγ-VF membranes (Fig. 2c). The two genotypes 134 \nexhibited comparable apparent dissociation constants (Kd = 13 nM for wild-type and 17 135 \nnM for ENaCγ-VF). These values indicate that the C-terminal mVenus-3C-3xFLAG tag 136 \ndoes not measurably alter benzamil affinity. The physiological and biochemical data 137 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 8 \nestablish that the tagged γ subunit preserves native ENaC function and 138 \npharmacological properties. 139 \n 140 \nDetection and characterization of native ENaC complexes using FSEC and 141 \nSiMPull 142 \nThe mVenus tag provides a sensitive fluorescence handle for tracking γ-containing 143 \nENaC assemblies in tissue lysates using both ensemble and single-molecule 144 \napproaches (Fig. 3a). FSEC48 separates protein complexes by size under non-145 \ndenaturing conditions and uses inline fluorescence to monitor tagged species as they 146 \nelute from a calibrated gel-filtration column. Originally developed for recombinant 147 \nmembrane proteins, FSEC has proven effective for assessing complex formation, 148 \nsample homogeneity, and expression levels48, 58. Here, we extend this approach to 149 \nnative tissues by using mVenus as an intrinsic reporter for γ-containing ENaC 150 \ncomplexes. 151 \n 152 \nWe applied FSEC to characterize native ENaC assemblies in lung, kidney, and colon 153 \ntissues from ENaCγ-VF mice. Lysates were fractionated on a Superose 6 Increase 154 \ncolumn, and mVenus fluorescence was monitored across the elution profile (Fig. 3a). 155 \nLung and kidney lysates displayed well-defined fluorescence peaks eluting between 11 156 \nand 16 mL, consistent with the expected size range of multimeric ENaC complexes (Fig. 157 \n3b). In contrast, colon lysates produced substantially lower signal in initial FSEC runs. 158 \nFree mVenus eluted at a later volume, allowing us to distinguish intact ENaCγ-159 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 9 \ncontaining complexes from unincorporated fluorophore and to verify sample integrity 160 \nprior to downstream analyses. 161 \n 162 \nTo complement ensemble analyses and resolve the ENaC composition at the single-163 \ncomplex level, we integrated SiMPull49. SiMPull combines immunoaffinity capture with 164 \ntotal internal reflection fluorescence (TIRF)59 microscopy to visualize individual protein 165 \ncomplexes under non-denaturing conditions. This approach is well suited for low-166 \nabundance membrane proteins such as ENaC, offering attomole-scale sensitivity60 167 \nwhile preserving native subunit interactions. For selective capture of ENaCγ-VF, we 168 \nemployed a biotinylated anti-GFP/mVenus nanobody61 that binds to the mVenus 169 \nfluorophore and enables immobilization of γ-containing assemblies on PEG-passivated, 170 \nstreptavidin-coated glass surfaces. 171 \n 172 \nFSEC was used not only as an ensemble characterization but also as a preparative 173 \nstep to ensure that single-molecule measurements interrogated intact complexes. 174 \nFractions eluting before free mVenus, corresponding to high-molecular-weight γ-175 \ncontaining assemblies, were selected for SiMPull. Using this FSEC-guided workflow, 176 \nrobust mVenus signals were detected in lung and kidney samples, confirming efficient 177 \nand specific capture of γ-containing complexes (Fig. 3c, d). For colon samples, where 178 \nFSEC indicated lower ENaC abundance, lysates were concentrated prior to SiMPull, 179 \nwhich enabled reliable single-molecule detection. 180 \n 181 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 10 \nThe combined use of FSEC and SiMPull provides a powerful, complementary platform 182 \nfor characterizing native ENaC assemblies. FSEC offers a rapid assessment of complex 183 \nintegrity and abundance, while SiMPull resolves individual complexes with single-184 \nmolecule precision. These approaches establish a robust analytical platform for 185 \nstudying ENaC composition and assembly across multiple epithelial tissues. The 186 \nobserved tissue-dependent differences in ENaC abundance and apparent complex size 187 \nare consistent with known variations in baseline Na+ transport and ENaC dependence 188 \nacross lung, kidney, and colon, and may contribute to how each epithelium matches 189 \nchannel activity to its specific absorptive demands1-7, 40. 190 \n 191 \nDual-color FSEC using a fluorescent Fab against ENaCα 192 \nHaving established that ENaCγ-VF provides a reliable fluorescence handle for tracking 193 \nγ-containing assemblies across tissues, we next asked whether this platform could be 194 \nextended to resolve the composition of native ENaC complexes at the subunit level. 195 \nBecause the mVenus tag reports specifically on the γ subunit, the analyses above 196 \ndefine the behavior of γ-containing assemblies but do not directly address the 197 \nincorporation of other ENaC subunits into the same complexes. A key advantage of the 198 \nENaCγ-VF allele is that it provides a stable biochemical anchor from which additional 199 \nsubunits can be assessed. To expand the detection capabilities of the system and 200 \nenable subunit-resolved interrogation of native ENaC assemblies, we developed a 201 \nfluorescent Fab directed against the extracellular domain of ENaCα. 202 \n 203 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 11 \nThe parent monoclonal antibody, 7B1, was originally raised against human ENaCα35, 204 \nand sequence alignment revealed conservation between human and mouse α subunits 205 \nwithin the predicted epitope (Fig. 4a, b), supporting the feasibility of cross-species 206 \nrecognition. To generate a monovalent, fluorescent reagent suitable for biochemical and 207 \nbiophysical studies, we cloned the variable regions of the 7B1 heavy and light chains 208 \ninto secretion-optimized expression constructs and fused the heavy chain C terminus to 209 \nthe red fluorescent protein mScarlet-I62, yielding 7B1-mScarlet (Fig. 4c). The Fab was 210 \nsecreted efficiently from HEK293 cells and isolated from conditioned media. 211 \n 212 \nWe first validated 7B1-mScarlet in a recombinant system expressing mouse ENaC 213 \ncomposed of wild-type α and β subunits and a mVenus-tagged γ subunit, recapitulating 214 \nthe configuration of the ENaCγ-VF allele (Fig. S2a). Detergent-solubilized lysates were 215 \nincubated with 7B1-mScarlet and analyzed by FSEC. mVenus and mScarlet 216 \nfluorescence co-eluted in a single peak at the expected position for trimeric ENaC, 217 \nwhereas control samples lacking 7B1-mScarlet showed no mScarlet signal within this 218 \nelution window (Fig. S2b). These results indicate that 7B1-mScarlet binds specifically to 219 \nfolded ENaC complexes containing the α subunit. To assess whether 7B1 recognizes α  220 \nacross different maturation states, we performed surface biotinylation of cells 221 \nexpressing recombinant mouse ENaC, lysed them, purified ENaC via the γ tag, and 222 \nprobed the resulting surface and intracellular fractions with 7B1-mScarlet (Fig. S2c); 223 \nrobust signal was detected in both biotinylated and non-biotinylated pools, indicating 224 \nthat the Fab can bind α  in multiple maturation states rather than a single conformational 225 \nend point (Fig. S2d). 226 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 12 \n 227 \nWe next asked whether this Fab could detect ENaCα within native γ-containing 228 \ncomplexes isolated from tissue. Lung lysates from ENaCγ-VF mice were enriched for γ-229 \ncontaining assemblies by affinity capture through the C-terminal FLAG tag and eluted 230 \nunder non-denaturing conditions. Addition of 7B1-mScarlet to the eluted material 231 \nfollowed by FSEC analysis revealed co-elution of mVenus and mScarlet fluorescence 232 \n(Fig. 4d-f), demonstrating that α and γ subunits are incorporated into the same native 233 \ncomplexes. Strikingly, while mVenus fluorescence extended across a broader range (11-234 \n16 mL), the 7B1-mScarlet signal was confined to the 11-13 mL window. These data 235 \nestablish dual-color FSEC as a non-denaturing approach for resolving the subunit 236 \ncomposition of native γ-containing ENaC assemblies. The distinct behavior indicates 237 \nthat α and γ subunits partition into different subsets of γ-containing assemblies and 238 \nhighlights heterogeneity within the γ-containing population. The observations motivated 239 \nfurther investigation into whether γ-containing assemblies of differing apparent sizes 240 \nengage distinct regulatory protein complexes in vivo. 241 \n 242 \nENaCγ-containing complexes associate with regulatory proteins in vivo 243 \nThe heterogeneity revealed by dual-color FSEC raised the possibility that g-containing 244 \nassemblies of different apparent sizes represent distinct molecular states. The presence 245 \nof both a-containing complexes and broader g-containing assemblies is consistent with 246 \na model in which these complexes correspond to distinct maturation or stability 247 \ncheckpoints that tune ENaC expression14-25, 39-44. In this context, the 7B1-positive 248 \npopulation represents α-containing complexes enriched within a narrower, higher 249 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 13 \nmolecular weight fraction, whereas γ-based fluorescence reports a continuum of γ-250 \ncontaining assemblies likely spanning multiple stages of folding, assembly, and 251 \nregulatory interactions. To examine this possibility and extend our analysis beyond 252 \nENaC subunits alone, we turned to mass spectrometry to identify proteins that co-purify 253 \nwith γ-containing assemblies. 254 \n 255 \nLarge-scale affinity purification was performed from lung, kidney, and colon tissues of 256 \nENaCγ-VF mice using anti-FLAG enrichment followed by 3C protease cleavage to 257 \ngently release native complexes under non-denaturing conditions (Fig. 5a). Fractions 258 \ncorresponding to the mVenus fluorescence peak were collected and subjected to mass 259 \nspectrometry analysis (Fig. 5b). As expected, ENaCγ co-purified with ENaCα and β 260 \nsubunits (Fig. 5c), consistent with our dual-color FSEC data demonstrating α-γ co-261 \nassembly (Fig. 4f). Recovery of all three subunits confirms the integrity of the purified 262 \ncomplexes and demonstrates that the ENaCγ-VF tag preserves native channel 263 \nassembly. 264 \n 265 \nIn addition to the core ENaC subunits, mass spectrometry revealed several regulatory 266 \nproteins that associate with γ-containing assemblies. Notably, the E3 ubiquitin ligase 267 \nNedd4-243 was detected across multiple tissues. Nedd4-2 is a well-established regulator 268 \nof ENaC surface expression and turnover, mediating ubiquitin-dependent internalization 269 \nand degradation. Its presence suggests that a subset of γ-containing complexes 270 \nengages components of the ubiquitin regulatory pathway. We also detected the ER 271 \nchaperone calnexin, consistent with its established role in the folding and quality control 272 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 14 \nof multi-subunit membrane proteins63-65, including ENaC. Its presence suggests that 273 \nanother subset of γ-containing complexes engages components of the protein quality -274 \ncontrol pathway. These associations support a model in which γ participates both in fully 275 \nassembled ENaC channels and in regulatory-associated forms,  providing a molecular 276 \nbasis for the broader elution profile observed by FSEC. 277 \n 278 \nTo evaluate the specificity of these interactions, we performed identical affinity 279 \npurifications from wild-type tissues lacking the mVenus-3C-3xFLAG tag (Fig. S3). 280 \nNeither Nedd4-2 nor calnexin was detected in these control samples, indicating that 281 \ntheir presence in ENaCγ-VF purifications reflects specific co-purification with γ-282 \ncontaining complexes rather than nonspecific retention on the affinity resin. These 283 \nfindings broaden the scope of our analysis from subunit composition to the regulatory 284 \nenvironment surrounding native ENaC. The ENaCγ-VF allele enables the isolation of 285 \nboth assembled αβγ channels, as well as γ-associated intermediary or regulatory 286 \ncomplexes, linking biochemical heterogeneity observed by FSEC to defined molecular 287 \npathways that govern ENaC maturation, quality control, and turnover in vivo. 288 \n 289 \nDiscussion 290 \nThis study introduces the ENaCγ-VF knock-in mouse line as a physiologically faithful 291 \nplatform for dissecting the composition, assembly, and regulatory environment of native 292 \nENaC complexes across epithelial tissues. By combining endogenous fluorescent 293 \ntagging with orthogonal biochemical and single-molecule approaches, this system 294 \novercomes longstanding challenges posed by ENaC’s low abundance, membrane 295 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 15 \nlocalization, and complex subunit stoichiometry. Preservation of normal physiology in 296 \nhomozygous ENaCγ-VF mice demonstrates that the C-terminal mVenus-3C-3xFLAG 297 \nfusion maintains native channel function, providing confidence that downstream 298 \nbiochemical and structural analyses reflect bona fide ENaC biology. 299 \n 300 \nA notable strength of the ENaCγ-VF allele is its broad compatibility with both advanced 301 \nand routine biochemical and imaging workflows. Here, we demonstrate that the mVenus 302 \ntag enables sensitive detection by ensemble fluorescence, FSEC, confocal imaging, 303 \nand TIRF microscopy. More generally, mVenus and FLAG tags also support robust 304 \ndetection by standard immunoblotting using well-validated commercial antibodies, 305 \nmaking the mouse line readily compatible with widely-used biochemical analyses. In 306 \nparallel, the C-terminal 3xFLAG epitope supports affinity purification followed by native-307 \nstate elution using 3C protease. These features allow intact ENaC assemblies to be 308 \nisolated without disrupting subunit composition or regulatory interactions. Thus, the 309 \nENaCγ-VF mouse provides a practical and adaptable foundation for examining ENaC at 310 \nmolecular, cellular, and tissue scales. 311 \n 312 \nFSEC and SiMPull analyses reveal substantial heterogeneity among γ-containing ENaC 313 \ncomplexes across lung, kidney, and colon. FSEC profiles identify both well-defined 314 \ntrimeric assemblies and broader, higher-molecular-weight populations, while SiMPull 315 \nconfirms that γ-containing complexes can be isolated and visualized at the single-316 \nmolecule level under non-denaturing conditions. These findings indicate that ENaCγ 317 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 16 \nparticipates in multiple molecular states in vivo and underscore the utility of the ENaCγ-318 \nVF system for resolving this complexity directly. 319 \n 320 \nTo distinguish fully assembled channels from intermediate or regulatory states, we 321 \ndeveloped 7B1-mScarlet, a fluorescent Fab that recognizes ENaCα under mild 322 \nconditions. Dual-color FSEC showed that α-containing complexes elute within a 323 \nnarrower molecular range than γ-containing assemblies, suggesting preferential 324 \nincorporation of α into a more restricted subset of αβγ channels. Validation in 325 \nrecombinant and native systems confirmed that 7B1-mScarlet selectively binds folded α 326 \nwithin intact ENaC complexes. 327 \n 328 \nWithin this context, γ occupies a central position in the ENaC assembly landscape. 329 \nUnlike α, which appears to be selectively incorporated into a narrower population of 330 \ncomplexes, γ persists across multiple assembly and regulatory states, positioning it as a 331 \nmolecular hub for ENaC biogenesis and turnover. The broad elution range detected by 332 \nFSEC, the presence of γ in both trimeric channels and regulatory-associated species, 333 \nand its interaction with chaperones and ubiquitin ligases collectively suggest that γ 334 \nparticipates in multiple checkpoints along the channel’s biogenesis, quality control, and 335 \nturnover pathways. In contrast, α appears in a more restricted set of molecular contexts, 336 \nconsistent with its rate-limiting role in channel assembly and preferential incorporation 337 \ninto fully assembled αβγ complexes. The sharp, 7B1-positive peak therefore most likely 338 \nreflects mature, fully assembled αβγ channels, whereas the broader γ profile 339 \nencompasses assembly intermediates and regulator -engaged species. This 340 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 17 \ninterpretation is in line with quantitative biochemical studies showing an excess of β 341 \nand γ over α subunits in native epithelia, implying that only a subset of γ participates in 342 \nfully assembled channels at any given time 32. These observations align with long-343 \nstanding physiological models in which differences in subunit abundance govern ENaC 344 \nassembly efficiency, but here are supported by direct biochemical analysis of intact 345 \nnative complexes. These findings indicate that native ENaC exists not as a single, static 346 \nentity but as an ensemble of molecular species that differ in subunit composition, 347 \nassembly state, and regulatory interactions. Decades of physiological and 348 \nelectrophysiological studies inferred such heterogeneity indirectly, but the lack of tools 349 \nto visualize and isolate native complexes obscured the underlying molecular 350 \narchitecture66. The ENaCγ-VF model, particularly when combined with subunit-specific 351 \nprobes such as 7B1-mScarlet and native-state purification, begins to resolve this 352 \ncomplexity with a level of precision that was previously inaccessible. 353 \n 354 \nMass spectrometry further supports this view by identifying regulatory factors that co-355 \npurify with γ-containing assemblies. In addition to α and β subunits, we detected Nedd4-356 \n2 and calnexin, key regulators of ENaC maturation, trafficking, and turnover. Their 357 \nselective recovery in ENaCγ-VF purifications, demonstrates that affinity isolation via the 358 \nγ subunit specifically enriches bona fide γ-associated ENaC complexes rather than 359 \nnonspecific background proteins. The identification of Nedd4-2 and calnexin within 360 \nthese purifications further indicates that a subset of γ-containing assemblies engages 361 \nquality-control machinery and regulatory pathways in vivo. This association provides a 362 \nmolecular explanation for the broader elution profile of γ-containing species and 363 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 18 \nhighlight the dynamic equilibrium between mature channels, assembly intermediates, 364 \nand regulatory states in vivo. Taken together with the preserved physiology of ENaCγ-365 \nVF mice and the specific recovery of known ENaC regulators, these observations 366 \nargue that the γ-containing species primarily reflect native assembly and quality -control 367 \nstates rather than artifacts of the γ-tag. 368 \n 369 \nMore broadly, the ENaCγ-VF platform enables a transition from indirect inference to 370 \ndirect molecular definition of native ENaC assemblies. By enabling isolation, 371 \nvisualization, and quantification of intact ENaC complexes under non-denaturing 372 \nconditions, this system provides a foundation for linking subunit composition and 373 \nregulatory state to tissue-specific ENaC function. As complementary tools for detecting 374 \nadditional ENaC subunits, particularly β, and for structural analysis of native complexes 375 \ncontinue to emerge, the ENaCγ-VF model will serve as a foundational resource for 376 \ndefining the molecular principles that govern ENaC regulation across epithelial systems. 377 \nIn future work, this platform can be applied to examine how disease states remodel 378 \nENaC assembly and regulatory interactions, linking molecular architecture to disordered 379 \nNa⁺ handling in conditions such as salt-sensitive hypertension, cystic fibrosis, and 380 \npseudohypoaldosteronism. 381 \nMaterials and Methods 382 \nGeneration of ENaCγ-VF mice 383 \nThe ENaCγ-VF knock-in mouse line was generated by the Jackson Laboratory using 384 \nCRISPR/Cas9-mediated genome editing to insert a sequence encoding mVenus, a 3C 385 \nprotease cleavage site, and a 3xFLAG tag at the C-terminus of the endogenous Scnn1g 386 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 19 \ngene. Heterozygous animals (ENaCγ-VF/+) were initially shipped to our lab for 387 \nvalidation. Following phenotypic and localization analysis (see Results), the Jackson 388 \nLaboratory provided homozygous (ENaCγ-VF/ENaCγ-VF) mice, which were also viable 389 \nand showed normal ENaCγ localization. Both heterozygous and homozygous mice are 390 \nmaintained under standard breeding protocols. To ensure continued integrity of the 391 \ntagged allele, we routinely verify the line using genotyping services with primers 392 \ndesigned to detect the presence of the mVenus-3xFLAG insertion. All animal 393 \nprocedures were conducted in accordance with protocols approved by the IACUC. 394 \nImmunofluorescence Microscopy 395 \nMice were anesthetized with a ketamine-xylazine-acepromazine cocktail (50:5:0.5 396 \nmg/kg). The kidneys were perfusion fixed by retrograde abdominal aortic perfusion of 397 \n3% paraformaldehyde in PBS (pH 7.4). After perfusion, the kidneys were removed, 398 \ndissected, and cryopreserved in 800 mOsm/L sucrose in PBS overnight before being 399 \nembedded in Tissue-Tek Optimal Cutting Temperature compound (Sakura Finetek, 400 \nTorrance, CA). Slides were prepared by cutting 5 mm sections and stored at -80°C until 401 \nuse. For imaging, slides were incubated with 0.5% Triton X-100 in PBS for 30 minutes, 402 \nblocked with 5% milk in PBS for 30 minutes, followed by incubation with primary 403 \nantibody, diluted in blocking buffer, for 1 hour at room temperature or overnight at 4°C. 404 \nSections were then washed with PBS three times and incubated with fluorescent dye-405 \nconjugated secondary antibody, diluted in blocking buffer, for 1 hour at room 406 \ntemperature. Sections were washed with PBS three times and stained with 4’,6-407 \ndiamidino-2-phenylindole before being mounted with ProLong Diamond Antifade 408 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 20 \nMountant (ThermoFisher Scientific, Carlsbad, CA). Images were captured using a 409 \nKEYENCE BZ-X800 microscope (Itasca, IL). 410 \n 411 \nAmiloride treatment and urine analysis 412 \nMice were injected intraperitoneally with vehicle (0.9% saline) and then placed in 413 \nmetabolic cages for a 6-hour urine collection. Five days later, the same animals 414 \nreceived intraperitoneal injection of amiloride hydrochloride (40 μg per 25 g body 415 \nweight), followed by another 6-hour urine collection. Sodium and potassium 416 \nconcentrations were measured by flame photometry, and the urinary Na⁺:K⁺ ratio was 417 \nused to assess ENaC function. 418 \nFilter binding assay 419 \nKidney tissue was dissected from adult wild-type or ENaCγ-VF mice, rinsed in ice-cold 420 \ntris buffered saline (TBS, 20mM Tris-HCl pH 7.6, 200mM NaCl), stored in TBS with 421 \nprotease inihibitors (Thermo Scientific™ Pierce protease inhibitor tablets, ThermoFisher 422 \nScientific) and flash-frozen in liquid nitrogen. For membrane preparation, frozen kidneys 423 \nwere pulverized using a liquid nitrogen-cooled mortar and pestle until a fine powder was 424 \nobtained. Tissue was then homogenized in homogenization buffer (50mM phosphate 425 \nbuffer, pH 7.5) using a Dounce homogenizer. The homogenate was centrifuged at 426 \n1,800g for 10 min at 4 °C to remove nuclei and cellular debris. The resulting 427 \nsupernatant was further centrifuged at 100,000g for 60 min at 4 °C to pellet membrane 428 \nfractions. Membrane pellets were resuspended in binding buffer (50mM phosphate 429 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 21 \nbuffer, pH 7.5). Equilibrium ligand binding was assessed using tritiated benzamil ([³H]-430 \nbenzamil; Moravek). Membrane suspensions (typically 10 mg) were incubated with 431 \nincreasing concentrations of [³H]-benzamil (0.5-300 nM) in a final volume of 500 µL 432 \nbinding buffer. Incubations were carried out at 22 °C for 2 hours, a duration sufficient to 433 \nreach equilibrium under these conditions. Nonspecific binding was determined in 434 \nparallel samples containing 100 µM unlabeled phenamil (Phenamil mesylate, Tocris 435 \nBioscience). Binding reactions were terminated by rapid vacuum filtration onto 436 \npresoaked glass microfiber filters (GF/B 25mm, Whatman) using a 12-well filtration 437 \nmanifold (Millipore). Filters were prewashed with 2ml ice-cold binding buffer and treated 438 \nwith 0.3% polyethyleneimine (PEI, MW 25K, Polysciences) to reduce nonspecific 439 \nradioligand adsorption. Incubated samples were applied and each well was washed 440 \nimmediately with 2 x 5 mL ice-cold binding buffer. Filters were dried by vacuum, 441 \ntransferred to scintillation vials, extracted in 5 mL scintillation cocktail (Ultima Gold), and 442 \nradioactivity was quantified by liquid scintillation counting (Beckman Coulter LS6500). 443 \nSpecific binding was calculated as total minus nonspecific counts per minute (CPM). 444 \nEquilibrium binding curves were fitted to a one-site binding model using nonlinear 445 \nregression in GraphPad Prism: 446 \n𝑌 = 𝐵max 𝑋\n𝐾! + 𝑋\t448 \n 447 \nwhere Y is specific binding, X is the free ligand concentration, Bₘₐₓ is maximal binding, 449 \nand Kd is the apparent dissociation constant. Data from ENaCγ-VF and wild-type 450 \nsamples were fitted independently, and similar Kd values were interpreted as evidence 451 \nthat the γ-tag does not perturb benzamil affinity. 452 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 22 \nTissue preparation and lysate generation for FSEC analysis 453 \nTissues were harvested from ENaCγ-VF mice. For each FSEC experiment, one lung (all 454 \nlobes), one kidney, and two colons were collected, rinsed in cold TBS, and weighed. 455 \nEach tissue was homogenized in solubilization buffer containing 100 mM Tris-HCl (pH 456 \n7.6), 200 mM NaCl, 1% n-dodecyl-β-D-maltoside (DDM, Anagrade, Anatrace), protease 457 \ninhibitors and Pefabloc® SC (Millipore Sigma). The homogenization volume was 458 \nstandardized to 750 µL per 100 mg of tissue. Samples were solubilized at 4 °C for 60 459 \nminutes with gentle rotation, then clarified by centrifugation at 100,000g twice for 40 460 \nminutes each. Supernatants were concentrated using a 100kDa centrifugal concentrator 461 \nto a final volume of 110 - 480 µL. Concentrated samples were then centrifuged again at 462 \n100,000g for 10 minutes to remove aggregates. The final cleared supernatant was 463 \ninjected onto a Superose 6 Increase column (Cytiva) for FSEC analysis (Waters 464 \nHPLC)48. Fractions were collected and analyzed based on mVenus fluorescence. 465 \nSiMPull Assays 466 \nGlass coverslips were cleaned and PEGylated using standard silanization procedures49, 467 \nthen functionalized sequentially with streptavidin and a biotinylated anti-GFP/mVenus 468 \nnanobody67. One experimental channel included the nanobody to capture fluorescently 469 \ntagged ENaC subunits, while a parallel channel without nanobody was used to quantify 470 \nbackground signal. 471 \nStreptavidin was applied at a concentration of 250 µg/mL and incubated for 5 minutes at 472 \nroom temperature, followed by a wash with the assay buffer (20 mM Tris-HCl (pH 7.6), 473 \n200 mM NaCl, 0.025% n-dodecyl-β-D-maltoside (DDM)) containing 200 µg/mL BSA. 474 \nBiotinylated nanobody was then applied at 3 µg/mL and incubated for 10 minutes, 475 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 23 \nfollowed again by a wash to remove unbound reagent. Clarified cell lysates (prepared 476 \nas for FSEC analysis) containing fluorescently tagged ENaC subunits were then applied 477 \nand incubated for 10 minutes at room temperature, followed by a final wash before 478 \nimaging. Chambers were imaged on a Leica DMi8 TIRF microscope with a 100x oil-479 \nimmersion objective. Images were captured using an Andor iXon Ultra 888 back-480 \nilluminated EMCCD camera with a 133 x 133 µm imaging area and 130 nm pixel size. 481 \nFor counting fluorophore spots, images were acquired using the excitation wavelength 482 \n488 nm. Spot detection and localization were performed using the ComDet plugin in 483 \nImageJ (FIJI). Molecule positions were analyzed within the center area of the field of 484 \nview (512x512 px). The approximate particle size was set at 5 pixels. The spot intensity 485 \nthreshold (in SD) was set to 3. Data were statistically analyzed using GraphPad Prism. 486 \n 487 \nAffinity Isolation of Native ENaC Complexes for FSEC and Mass Spectrometry 488 \nTissue preparation for this assay was performed using the same protocol as described 489 \nfor FSEC analysis, with the exception that digitonin was used as the detergent for 490 \nsolubilization instead of DDM. Briefly, lungs, kidneys, and colons were collected from 5 491 \nmice, and homogenized in solubilization buffer containing 100 mM Tris-HCl pH 7.6, 200 492 \nmM NaCl, 5mM EDTA, 1% digitonin (High purity, Millipore Sigma), protease inhibitors 493 \nand Pefabloc. Clarified lysates were incubated with 50 µL of anti-FLAG M2 magnetic 494 \nbeads (Sigma-Aldrich) overnight at 4 °C with gentle rotation. Beads were washed 10 495 \ntimes with 15 ml wash buffer (20 mM Tris-HCl pH 7.6, 200 mM NaCl, 0.1% digitonin), 496 \nand proteins were eluted by incubating with 25 µg of PreScission Proteases (GenScript) 497 \nfor 6 hours at 4 °C. The eluted material, in a total volume of 400 uL, was concentrated 498 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 24 \nusing centrifugal filters prior to injection onto a Superose 6 Increase 10/300 column 499 \n(Cytiva) equilibrated in FSEC buffer (20 mM Tris-HCl pH 7.6, 200 mM NaCl, 0.1% 500 \ndigitonin). Fluorescence was monitored using an inline fluorimeter (Shimadzu RF-20A) 501 \nto track mVenus-tagged ENaCγ. Fractions corresponding to the fluorescent signal were 502 \ncollected, pooled, and concentrated again for downstream mass spectrometry analysis. 503 \n 504 \n7B1-mScarlet Fab Production 505 \nDNA sequences encoding the heavy and light chains of the Fab fragment of the anti-506 \nENaCα monoclonal antibody 7B1 were cloned into separate pEG BacMam expression 507 \nplasmids68. The heavy chain construct was engineered to include mScarlet-I at the C-508 \nterminus, followed by a Strep-tag II for affinity purification. Both constructs included an 509 \nN-terminal leader sequence (MGWSCIILFLVATATGVHS) that allows for secretion of the 510 \nFab into the media. First passage of mBaculovirus was generated from each plasmid 511 \nusing Sf9 insect cells cultured at a density of 0.5x10⁶ cells/mL at 27 °C for 5 days. P1 512 \nvirus was used to reinfect Sf9 insect cells cultured at a density of 1x10⁶ cells/mL at 513 \n27 °C for 4 days.  After incubation, cells were pelleted by centrifugation at 1,800g for 20 514 \nminutes, and the supernatant containing the virus was harvested and filtered through a 515 \n0.22 µm filter. The resulting virus was used to infect suspension-adapted HEK293 cells 516 \nat a density of 3x10⁶ cells/mL and incubated at 37 °C for 8 hours. After 8 hours, 10mM 517 \nsodium butyrate was added and the cells were moved to a 30 °C incubator. Cells were 518 \ncultured at 30 °C for 96 hours, after which they were spun down and the culture 519 \nsupernatant was collected and filtered again using a 0.22 µm filter. The clarified media 520 \nwith BioLock (1.6ml/L of FreeStyle™ 293, IBA Lifesciences) was loaded onto a 10 mL 521 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 25 \nStrep-Tactin affinity resin (5ml/1L of media) (Cytiva) pre-equilibrated with wash buffer 522 \n(20mM Tris pH 7.6, 200mM NaCl and 0.025% n-dodecyl-β-D-maltoside (DDM)). The 523 \ncolumn was washed with 20 column volumes of wash buffer, and bound 7B1-mScarlet 524 \nFab was eluted using desthiobiotin-containing wash buffer. Desthiobiotin (IBA 525 \nLifesciences) was subsequently removed using a PD-10 desalting column (Cytiva), and 526 \nthe Fab was eluted in wash buffer. Purity and labeling of the Fab were confirmed by 527 \nSDS-PAGE and FSEC. 528 \n 529 \nCell-surface biotinylation and validation of 7B1-mScarlet binding to recombinant 530 \nENaC 531 \nTo evaluate binding of the 7B1-mScarlet Fab to recombinant mouse ENaC, we 532 \nexpressed a mouse ENaC construct composed of the α (Uniprot ID Q61180), β (Uniprot 533 \nID Q9WU38), and γ (Uniprot ID Q9WU39) subunits, with the γ subunit bearing the C-534 \nterminal mVenus tag. Cells were analyzed under both total and plasma membrane-535 \nenriched conditions. For total protein analysis, cells were solubilized directly36, 37, 536 \nclarified by centrifugation, and the resulting lysates were incubated with 7B1-mScarlet 537 \nprior to downstream analysis. To specifically assess plasma membrane-expressed 538 \nENaC, cell-surface proteins were labeled using a membrane-impermeant biotinylation 539 \nreagent according to the manufacturer’s instructions (Pierce™ Cell surface biotinylation 540 \n& Isolation Kit, ThermoFisher Scientific). Briefly, cells expressing recombinant mouse 541 \nENaC were harvested and incubated with biotin (EZ-Link Sulfo-NHS-SS-Biotin) to 542 \nselectively label surface-exposed proteins. Excess biotin was quenched and removed 543 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 26 \nby washing, after which cells were either flash-frozen for storage at -80 °C or processed 544 \nimmediately for solubilization. 545 \n 546 \nCells were lysed in buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 20mM 547 \nDDM, 3mM CHS, and protease inhibitors. Lysates were clarified by centrifugation, and 548 \nthe supernatant was incubated with GFP nanobody resin (GNB). Following binding, the 549 \nresin was washed with 20mM Tris (pH7.6), 150mM NaCl, 0.5mM DDM, 75uM CHS 550 \nwithout and with 5mM CaCl2.  Both biotinylated and non-biotinylated fractions of the 551 \nprotein were eluted by thrombin (30ug/ml of resin, Human alpha-thrombin, Prolytix) 552 \ncleavage. The thrombin eluted sample was passed through the Strep-Tactin resin to 553 \ncapture biotinylated, plasma membrane-expressed proteins. The unbound flow-554 \nthrough, representing the intracellular protein fraction, was collected. Following 555 \nfractionation, 7B1-mScarlet was added to the flow-through fraction. Biotinylated proteins 556 \nbound to the Strep-Tactin resin were subsequently eluted using excess biotin (10x 557 \nBuffer BXT, IBA Lifesciences), and 7B1-mScarlet was added to the eluted plasma 558 \nmembrane fraction. Both intracellular (flow-through) and plasma membrane (biotin-559 \neluted) fractions were analyzed by HPLC, with fluorescence monitored in 560 \nthe mScarlet channel.  561 \nData Availability Statement 562 \nAll data in this manuscript are available from the corresponding author upon request. 563 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 27 \nAcknowledgements 564 \nWe thank members of the Gouaux laboratory, especially Eric Gouaux, April Goehring, 565 \nand Natalie Sheldon, for their generous guidance and support as we established 566 \nanimal-based experiments and native-state biochemical workflows. We are grateful to 567 \nAshok Reddy and the staff of the OHSU Proteomics Shared Resource for mass 568 \nspectrometry analyses and technical expertise. We thank the OHSU Department of 569 \nComparative Medicine for assistance with animal care and colony management. The 570 \nENaCγ-VF mouse line was generated by The Jackson Laboratory. Funding for this 571 \nproject was provided by institutional support from the Vollum Institute and by NIH grant 572 \nR01GM138862 to I.B, R01DK132066 to J.A.M, and R01s DK133220 and DK51496 to 573 \nD.E. 574 \n 575 \nAuthor Contributions 576 \nA.B. maintained the mouse colony, harvested mouse tissues, and performed FSEC, 577 \nSiMPull, and purification of native ENaC complexes. J.C. performed the amiloride 578 \nchallenge experiments. X.-T.S. conducted confocal imaging of heterozygous and 579 \nhomozygous mice. R.B.M. maintained the mouse colony and harvested mouse tissues. 580 \nJ.A.M. and D.H.E. supervised the physiology experiments. I.B. conceived and 581 \nsupervised the study. All authors contributed to manuscript preparation. 582 \n 583 \nDeclaration of interests 584 \nThe authors declare no competing interests. 585 \n 586 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 28 \nReferences 587 \n 588 \n1. Hamm LL, Feng Z, Hering-Smith KS. Regulation of sodium transport by ENaC in 589 \nthe kidney. Curr Opin Nephrol Hypertens. 2010;19(1):98-105. doi: 590 \n10.1097/MNH.0b013e328332bda4. PubMed PMID: 19996890; PMCID: PMC2895494. 591 \n2. Matalon S, Bartoszewski R, Collawn JF. Role of epithelial sodium channels in the 592 \nregulation of lung fluid homeostasis. Am J Physiol Lung Cell Mol Physiol. 593 \n2015;309(11):L1229-38. Epub 2015/10/04. doi: 10.1152/ajplung.00319.2015. PubMed 594 \nPMID: 26432872; PMCID: PMC4669342. 595 \n3. Rossier BC, Pradervand S, Schild L, Hummler E. Epithelial sodium channel and 596 \nthe control of sodium balance: interaction between genetic and environmental factors. 597 \nAnnu Rev Physiol. 2002;64:877-97. doi: 10.1146/annurev.physiol.64.082101.143243. 598 \nPubMed PMID: 11826291. 599 \n4. Hanukoglu I, Hanukoglu A. Epithelial sodium channel (ENaC) family: Phylogeny, 600 \nstructure-function, tissue distribution, and associated inherited diseases. Gene. 601 \n2016;579(2):95-132. Epub 2016/01/17. doi: 10.1016/j.gene.2015.12.061. PubMed 602 \nPMID: 26772908; PMCID: PMC4756657. 603 \n5. Matthay MA, Folkesson HG, Verkman AS. Salt and water transport across 604 \nalveolar and distal airway epithelia in the adult lung. Am J Physiol. 1996;270(4 Pt 605 \n1):L487-503. doi: 10.1152/ajplung.1996.270.4.L487. PubMed PMID: 8928808. 606 \n6. Gaillard D, Hinnrasky J, Coscoy S, Hofman P, Matthay MA, Puchelle E, Barbry P. 607 \nEarly expression of beta- and gamma-subunits of epithelial sodium channel during 608 \nhuman airway development. Am J Physiol Lung Cell Mol Physiol. 2000;278(1):L177-84. 609 \ndoi: 10.1152/ajplung.2000.278.1.L177. PubMed PMID: 10645905. 610 \n7. Malsure S, Wang Q, Charles RP, Sergi C, Perrier R, Christensen BM, Maillard M, 611 \nRossier BC, Hummler E. Colon-specific deletion of epithelial sodium channel causes 612 \nsodium loss and aldosterone resistance. J Am Soc Nephrol. 2014;25(7):1453-64. Epub 613 \n20140130. doi: 10.1681/ASN.2013090936. PubMed PMID: 24480829; PMCID: 614 \nPMC4073440. 615 \n8. Mall MA. ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR 616 \nmodulator therapies. Eur Respir J. 2020;56(6). Epub 20201224. doi: 617 \n10.1183/13993003.00946-2020. PubMed PMID: 32732328; PMCID: PMC7758539. 618 \n9. Chang SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild 619 \nL, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, Lifton RP. Mutations in subunits 620 \nof the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, 621 \npseudohypoaldosteronism type 1. Nat Genet. 1996;12(3):248-53. Epub 1996/03/01. doi: 622 \n10.1038/ng0396-248. PubMed PMID: 8589714. 623 \n10. Kerem E, Bistritzer T, Hanukoglu A, Hofmann T, Zhou Z, Bennett W, MacLaughlin 624 \nE, Barker P, Nash M, Quittell L, Boucher R, Knowles MR. Pulmonary epithelial sodium-625 \nchannel dysfunction and excess airway liquid in pseudohypoaldosteronism. N Engl J 626 \nMed. 1999;341(3):156-62. Epub 1999/07/15. doi: 10.1056/NEJM199907153410304. 627 \nPubMed PMID: 10403853. 628 \n11. Saxena A, Hanukoglu I, Saxena D, Thompson RJ, Gardiner RM, Hanukoglu A. 629 \nNovel mutations responsible for autosomal recessive multisystem 630 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 29 \npseudohypoaldosteronism and sequence variants in epithelial sodium channel alpha-, 631 \nbeta-, and gamma-subunit genes. J Clin Endocrinol Metab. 2002;87(7):3344-50. Epub 632 \n2002/07/11. doi: 10.1210/jcem.87.7.8674. PubMed PMID: 12107247. 633 \n12. Hummler E, Barker P, Talbot C, Wang Q, Verdumo C, Grubb B, Gatzy J, Burnier 634 \nM, Horisberger JD, Beermann F, Boucher R, Rossier BC. A mouse model for the renal 635 \nsalt-wasting syndrome pseudohypoaldosteronism. Proc Natl Acad Sci U S A. 636 \n1997;94(21):11710-5. doi: 10.1073/pnas.94.21.11710. PubMed PMID: 9326675; 637 \nPMCID: PMC23605. 638 \n13. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, 639 \nRossier BC. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-640 \ndeficient mice. Nat Genet. 1996;12(3):325-8. Epub 1996/03/01. doi: 10.1038/ng0396-641 \n325. PubMed PMID: 8589728. 642 \n14. Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams 643 \nC, Rossier BC, Lifton RP. A de novo missense mutation of the beta subunit of the 644 \nepithelial sodium channel causes hypertension and Liddle syndrome, identifying a 645 \nproline-rich segment critical for regulation of channel activity1995. PubMed PMID: 646 \n13006205075825840505related:eVF7TJtSf7QJ. 647 \n15. Bubien JK, Ismailov, II, Berdiev BK, Cornwell T, Lifton RP, Fuller CM, Achard JM, 648 \nBenos DJ, Warnock DG. Liddle's disease: abnormal regulation of amiloride-sensitive 649 \nNa+ channels by beta-subunit mutation. Am J Physiol. 1996;270(1 Pt 1):C208-13. Epub 650 \n1996/01/01. doi: 10.1152/ajpcell.1996.270.1.C208. PubMed PMID: 8772446. 651 \n16. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, 652 \nSchambelan M, Gill JR, Ulick S, Milora RV, Findling JW. Liddle&apos;s syndrome: 653 \nheritable human hypertension caused by mutations in the beta subunit of the epithelial 654 \nsodium channel. Cell. 1994;79(3):407-14. PubMed PMID: 7954808. 655 \n17. Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB, 656 \nWelsh MJ. Mechanism by which Liddle's syndrome mutations increase activity of a 657 \nhuman epithelial Na+ channel. Cell. 1995;83(6):969-78. doi: 10.1016/0092-658 \n8674(95)90212-0. PubMed PMID: 8521520. 659 \n18. Schild L, Lu Y, Gautschi I, Schneeberger E, al e. Identification of a PY motif in the 660 \nepithelial Na channel subunits as a target sequence for mutations causing channel 661 \nactivation found in Liddle syndrome. The EMBO  …. 1996. PubMed PMID: C6CE859B-662 \n6F7D-4304-900D-D9D35CAA7367. 663 \n19. Warnock DG, Bubien JK. Liddle syndrome: clinical and cellular abnormalities. 664 \nHosp Pract (Off Ed). 1994;29(7):95-8, 104-5. Epub 1994/07/15. PubMed PMID: 665 \n8027210. 666 \n20. Van Huysse JW, Amin MS, Yang B, Leenen FHH. Salt-induced hypertension in a 667 \nmouse model of Liddle syndrome is mediated by epithelial sodium channels in the brain. 668 \nHypertension. 2012;60(3):691-6. doi: 10.1161/HYPERTENSIONAHA.112.193045. 669 \nPubMed PMID: 22802227; PMCID: PMC3514876. 670 \n21. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa 671 \nC, Iwasaki T, Rossier B, Lifton RP. Hypertension caused by a truncated epithelial 672 \nsodium channel γ subunit: genetic heterogeneity of Liddle syndrome. Nature Genetics. 673 \n1995;11(1):76-82. doi: 10.1038/ng0995-76. PubMed PMID: 674 \n3059133470290364987related:O0oq6a45dCoJ. 675 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 30 \n22. Sun Y, Zhang J-n, Zhao D, Wang Q-s, Gu Y-c, Ma H-p, Zhang Z-r. Role of the 676 \nepithelial sodium channel in salt-sensitive hypertension. Acta Pharmacologica Sinica. 677 \n2011;32(6):789-97. doi: 10.1038/aps.2011.72. 678 \n23. Pitzer AL, Van Beusecum JP, Kleyman TR, Kirabo A. ENaC in Salt-Sensitive 679 \nHypertension: Kidney and Beyond. Curr Hypertens Rep. 2020;22(9):69. Epub 680 \n20200827. doi: 10.1007/s11906-020-01067-9. PubMed PMID: 32852643; PMCID: 681 \nPMC7452925. 682 \n24. Takahashi H, Yoshika M, Komiyama Y, Nishimura M. The central mechanism 683 \nunderlying hypertension: a review of the roles of sodium ions, epithelial sodium 684 \nchannels, the renin&ndash;angiotensin&ndash;aldosterone system, oxidative stress and 685 \nendogenous digitalis in the brain. Hypertension research : official journal of the 686 \nJapanese Society of Hypertension. 2011;34(11):1147-60. doi: 10.1038/hr.2011.105. 687 \nPubMed PMID: 21814209. 688 \n25. Gudmundsdottir H, Hoieggen A, Stenehjem A, Waldum B, Os I. Hypertension in 689 \nwomen: latest findings and clinical implications. Ther Adv Chronic Dis. 2012;3(3):137-690 \n46. doi: 10.1177/2040622312438935. PubMed PMID: 23251774; PMCID: 691 \nPMC3513905. 692 \n26. Lim SS, Vos T, Flaxman AD, Danaei G, Shibuya K, Adair-Rohani H, Amann M, 693 \nAnderson HR, Andrews KG, Aryee M, Atkinson C, Bacchus LJ, Bahalim AN, 694 \nBalakrishnan K, Balmes J, Barker-Collo S, Baxter A, Bell ML, Blore JD, Blyth F, Bonner 695 \nC, Borges G, Bourne R, Boussinesq M, Brauer M, Brooks P, Bruce NG, Brunekreef B, 696 \nBryan-Hancock C, Bucello C, Buchbinder R, Bull F, Burnett RT, Byers TE, Calabria B, 697 \nCarapetis J, Carnahan E, Chafe Z, Charlson F, Chen H, Chen JS, Cheng AT-A, Child 698 \nJC, Cohen A, Colson KE, Cowie BC, Darby S, Darling S, Davis A, Degenhardt L, 699 \nDentener F, Des Jarlais DC, Devries K, Dherani M, Ding EL, Dorsey ER, Driscoll T, 700 \nEdmond K, Ali SE, Engell RE, Erwin PJ, Fahimi S, Falder G, Farzadfar F, Ferrari A, 701 \nFinucane MM, Flaxman S, Fowkes FGR, Freedman G, Freeman MK, Gakidou E, 702 \nGhosh S, Giovannucci E, Gmel G, Graham K, Grainger R, Grant B, Gunnell D, 703 \nGutierrez HR, Hall W, Hoek HW, Hogan A, Hosgood HD, Hoy D, Hu H, Hubbell BJ, 704 \nHutchings SJ, Ibeanusi SE, Jacklyn GL, Jasrasaria R, Jonas JB, Kan H, Kanis JA, 705 \nKassebaum N, Kawakami N, Khang Y-H, Khatibzadeh S, Khoo J-P, Kok C, Laden F, 706 \nLalloo R, Lan Q, Lathlean T, Leasher JL, Leigh J, Li Y, Lin JK, Lipshultz SE, London S, 707 \nLozano R, Lu Y, Mak J, Malekzadeh R, Mallinger L, Marcenes W, March L, Marks R, 708 \nMartin R, McGale P, McGrath J, Mehta S, Mensah GA, Merriman TR, Micha R, Michaud 709 \nC, Mishra V, Mohd Hanafiah K, Mokdad AA, Morawska L, Mozaffarian D, Murphy T, 710 \nNaghavi M, Neal B, Nelson PK, Nolla JM, Norman R, Olives C, Omer SB, Orchard J, 711 \nOsborne R, Ostro B, Page A, Pandey KD, Parry CDH, Passmore E, Patra J, Pearce N, 712 \nPelizzari PM, Petzold M, Phillips MR, Pope D, Pope CA, Powles J, Rao M, Razavi H, 713 \nRehfuess EA, Rehm JT, Ritz B, Rivara FP, Roberts T, Robinson C, Rodriguez-Portales 714 \nJA, Romieu I, Room R, Rosenfeld LC, Roy A, Rushton L, Salomon JA, Sampson U, 715 \nSanchez-Riera L, Sanman E, Sapkota A, Seedat S, Shi P, Shield K, Shivakoti R, Singh 716 \nGM, Sleet DA, Smith E, Smith KR, Stapelberg NJC, Steenland K, Stöckl H, Stovner LJ, 717 \nStraif K, Straney L, Thurston GD, Tran JH, Van Dingenen R, van Donkelaar A, Veerman 718 \nJL, Vijayakumar L, Weintraub R, Weissman MM, White RA, Whiteford H, Wiersma ST, 719 \nWilkinson JD, Williams HC, Williams W, Wilson N, Woolf AD, Yip P, Zielinski JM, Lopez 720 \nAD, Murray CJL, Ezzati M, AlMazroa MA, Memish ZA. A comparative risk assessment 721 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 31 \nof burden of disease and injury attributable to 67 risk factors and risk factor clusters in 722 \n21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 723 \n2010. Lancet (London, England). 2012;380(9859):2224-60. doi: 10.1016/S0140-724 \n6736(12)61766-8. PubMed PMID: 23245609; PMCID: PMC4156511. 725 \n27. Whelton PK, Carey RM, Aronow WS, Casey DE, Jr., Collins KJ, Dennison 726 \nHimmelfarb C, DePalma SM, Gidding S, Jamerson KA, Jones DW, MacLaughlin EJ, 727 \nMuntner P, Ovbiagele B, Smith SC, Jr., Spencer CC, Stafford RS, Taler SJ, Thomas RJ, 728 \nWilliams KA, Sr., Williamson JD, Wright JT, Jr. 2017 729 \nACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the 730 \nPrevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A 731 \nReport of the American College of Cardiology/American Heart Association Task Force 732 \non Clinical Practice Guidelines. Hypertension. 2018;71(6):e13-e115. Epub 20171113. 733 \ndoi: 10.1161/HYP.0000000000000065. PubMed PMID: 29133356. 734 \n28. Xu J, Murphy SL, Kochanek KD, Arias E. Mortality in the United States, 2021. 735 \nNCHS Data Brief. 2022(456):1-8. PubMed PMID: 36598387. 736 \n29. Burnier M, Damianaki A. Hypertension as Cardiovascular Risk Factor in Chronic 737 \nKidney Disease. Circ Res. 2023;132(8):1050-63. Epub 20230413. doi: 738 \n10.1161/CIRCRESAHA.122.321762. PubMed PMID: 37053276. 739 \n30. Pacholko A, Iadecola C. Hypertension, Neurodegeneration, and Cognitive 740 \nDecline. Hypertension. 2024;81(5):991-1007. Epub 20240301. doi: 741 \n10.1161/HYPERTENSIONAHA.123.21356. PubMed PMID: 38426329; PMCID: 742 \nPMC11023809. 743 \n31. Sariban-Sohraby S, Benos DJ. The amiloride-sensitive sodium channel. Am J 744 \nPhysiol. 1986;250(2 Pt 1):C175-90. doi: 10.1152/ajpcell.1986.250.2.C175. PubMed 745 \nPMID: 2420186. 746 \n32. Frindt G, Meyerson JR, Satty A, Scandura JM, Palmer LG. Expression of ENaC 747 \nsubunits in epithelia. J Gen Physiol. 2022;154(10). Epub 20220808. doi: 748 \n10.1085/jgp.202213124. PubMed PMID: 35939271; PMCID: PMC9387651. 749 \n33. Shehata MF. The Epithelial Sodium Channel alpha subunit (alpha ENaC) 750 \nalternatively spliced form \"b\" in Dahl rats: What's next? Int Arch Med. 2010;3:14. Epub 751 \n20100706. doi: 10.1186/1755-7682-3-14. PubMed PMID: 20604958; PMCID: 752 \nPMC2909934. 753 \n34. Krueger B, Schlotzer-Schrehardt U, Haerteis S, Zenkel M, Chankiewitz VE, 754 \nAmann KU, Kruse FE, Korbmacher C. Four subunits (alphabetagammadelta) of the 755 \nepithelial sodium channel (ENaC) are expressed in the human eye in various locations. 756 \nInvest Ophthalmol Vis Sci. 2012;53(2):596-604. Epub 20120202. doi: 10.1167/iovs.11-757 \n8581. PubMed PMID: 22167092. 758 \n35. Noreng S, Bharadwaj A, Posert R, Yoshioka C, Baconguis I. Structure of the 759 \nhuman epithelial sodium channel by cryo-electron microscopy. Elife. 2018;7. Epub 760 \n2018/09/27. doi: 10.7554/eLife.39340. PubMed PMID: 30251954; PMCID: 761 \nPMC6197857. 762 \n36. Noreng S, Posert R, Bharadwaj A, Houser A, Baconguis I. Molecular principles of 763 \nassembly, activation, and inhibition in epithelial sodium channel. Elife. 2020;9. Epub 764 \n20200730. doi: 10.7554/eLife.59038. PubMed PMID: 32729833; PMCID: PMC7413742. 765 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 32 \n37. Houser A, Baconguis I. Structural insights into subunit-dependent functional 766 \nregulation in epithelial sodium channels. Structure. 2024. Epub 20241205. doi: 767 \n10.1016/j.str.2024.11.013. PubMed PMID: 39667931. 768 \n38. Fyfe GK, Canessa CM. Subunit composition determines the single channel 769 \nkinetics of the epithelial sodium channel. J Gen Physiol. 1998;112(4):423-32. Epub 770 \n1998/10/06. PubMed PMID: 9758861; PMCID: PMC2229421. 771 \n39. Kleyman TR, Carattino MD, Hughey RP. ENaC at the cutting edge: regulation of 772 \nepithelial sodium channels by proteases. J Biol Chem. 2009;284(31):20447-51. Epub 773 \n2009/04/30. doi: 10.1074/jbc.R800083200. PubMed PMID: 19401469; PMCID: 774 \nPMC2742807. 775 \n40. Kellenberger S, Schild L. Epithelial sodium channel/degenerin family of ion 776 \nchannels: a variety of functions for a shared structure. Physiological Reviews. 777 \n2002;82(3):735-67. doi: 10.1152/physrev.00007.2002. PubMed PMID: 12087134. 778 \n41. Rossier BC, Stutts MJ. Activation of the Epithelial Sodium Channel (ENaC) by 779 \nSerine Proteases. Annual Review of Physiology. 2009;71(1):361-79. doi: 780 \n10.1146/annurev.physiol.010908.163108. PubMed PMID: 18928407. 781 \n42. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, Boucher 782 \nRC. CFTR as a cAMP-dependent regulator of sodium channels. Science. 783 \n1995;269(5225):847-50. doi: 10.1126/science.7543698. PubMed PMID: 7543698. 784 \n43. Staub O, Abriel H, Plant P, Ishikawa T, Kanelis V, Saleki R, Horisberger JD, 785 \nSchild L, Rotin D. Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. 786 \nKidney Int. 2000;57(3):809-15. doi: 10.1046/j.1523-1755.2000.00919.x. PubMed PMID: 787 \n10720933. 788 \n44. Lang F, Shumilina E. Regulation of ion channels by the serum- and 789 \nglucocorticoid-inducible kinase SGK1. The FASEB Journal. 2013;27(1):3-12. doi: 790 \n10.1096/fj.12-218230. 791 \n45. Hill WG, An B, Johnson JP. Endogenously expressed epithelial sodium channel 792 \nis present in lipid rafts in A6 cells. J Biol Chem. 2002;277(37):33541-4. Epub 20020806. 793 \ndoi: 10.1074/jbc.C200309200. PubMed PMID: 12167633. 794 \n46. Hill WG, Butterworth MB, Wang H, Edinger RS, Lebowitz J, Peters KW, Frizzell 795 \nRA, Johnson JP. The epithelial sodium channel (ENaC) traffics to apical membrane in 796 \nlipid rafts in mouse cortical collecting duct cells. J Biol Chem. 2007;282(52):37402-11. 797 \nEpub 20071010. doi: 10.1074/jbc.M704084200. PubMed PMID: 17932048. 798 \n47. Rossier BC. Hormonal regulation of the epithelial sodium channel ENaC: N or 799 \nP(o)? J Gen Physiol. 2002;120(1):67-70. doi: 10.1085/jgp.20028638. PubMed PMID: 800 \n12084776; PMCID: PMC2311401. 801 \n48. Kawate T, Gouaux E. Fluorescence-detection size-exclusion chromatography for 802 \nprecrystallization screening of integral membrane proteins. Structure. 2006;14(4):673-803 \n81. doi: 10.1016/j.str.2006.01.013. PubMed PMID: 16615909. 804 \n49. Jain A, Liu R, Xiang YK, Ha T. Single-molecule pull-down for studying protein 805 \ninteractions. Nat Protoc. 2012;7(3):445-52. Epub 20120209. doi: 806 \n10.1038/nprot.2011.452. PubMed PMID: 22322217; PMCID: PMC3654178. 807 \n50. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A. A variant of 808 \nyellow fluorescent protein with fast and efficient maturation for cell-biological 809 \napplications. Nat Biotechnol. 2002;20(1):87-90. doi: 10.1038/nbt0102-87. PubMed 810 \nPMID: 11753368. 811 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 33 \n51. Cordingley MG, Callahan PL, Sardana VV, Garsky VM, Colonno RJ. Substrate 812 \nrequirements of human rhinovirus 3C protease for peptide cleavage in vitro. J Biol 813 \nChem. 1990;265(16):9062-5. PubMed PMID: 2160953. 814 \n52. Inc. N. Accelerate your research with the latest in computational biology 815 \nWilmington, Delaware2022. Available from: https://neurosnap.ai/. 816 \n53. Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X. 817 \nGeneration of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 818 \n2013;23(5):720-3. Epub 20130402. doi: 10.1038/cr.2013.46. PubMed PMID: 23545779; 819 \nPMCID: PMC3641603. 820 \n54. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. 821 \nOne-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-822 \nmediated genome engineering. Cell. 2013;153(4):910-8. Epub 20130502. doi: 823 \n10.1016/j.cell.2013.04.025. PubMed PMID: 23643243; PMCID: PMC3969854. 824 \n55. Barker PM, Nguyen MS, Gatzy JT, Grubb B, Norman H, Hummler E, Rossier B, 825 \nBoucher RC, Koller B. Role of gammaENaC subunit in lung liquid clearance and 826 \nelectrolyte balance in newborn mice. Insights into perinatal adaptation and 827 \npseudohypoaldosteronism. J Clin Invest. 1998;102(8):1634-40. doi: 10.1172/JCI3971. 828 \nPubMed PMID: 9788978; PMCID: PMC509015. 829 \n56. Picard N, Eladari D, El Moghrabi S, Planes C, Bourgeois S, Houillier P, Wang Q, 830 \nBurnier M, Deschenes G, Knepper MA, Meneton P, Chambrey R. Defective ENaC 831 \nprocessing and function in tissue kallikrein-deficient mice. J Biol Chem. 832 \n2008;283(8):4602-11. Epub 20071217. doi: 10.1074/jbc.M705664200. PubMed PMID: 833 \n18086683. 834 \n57. Kleyman TR, Cragoe EJ, Jr. Amiloride and its analogs as tools in the study of ion 835 \ntransport. J Membr Biol. 1988;105(1):1-21. Epub 1988/10/01. PubMed PMID: 2852254. 836 \n58. Hattori M, Hibbs RE, Gouaux E. A fluorescence-detection size-exclusion 837 \nchromatography-based thermostability assay for membrane protein precrystallization 838 \nscreening. Structure. 2012;20(8):1293-9. doi: 10.1016/j.str.2012.06.009. PubMed PMID: 839 \n22884106; PMCID: PMC3441139. 840 \n59. Axelrod D. Chapter 7: Total internal reflection fluorescence microscopy. Methods 841 \nCell Biol. 2008;89:169-221. doi: 10.1016/S0091-679X(08)00607-9. PubMed PMID: 842 \n19118676. 843 \n60. Clark S, Elferich J, Gai J, Goehring A, Mitra J, Ha T, Gouaux E. Strategy for 844 \nCompositional Analysis of the Hair Cell Mechanotransduction Complex Using TIRF 845 \nMicroscopy. Microsc Microanal. 2019;25(Suppl 2):1266-7. Epub 20190805. doi: 846 \n10.1017/s1431927619007062. PubMed PMID: 32025193; PMCID: PMC7001662. 847 \n61. Kubala MH, Kovtun O, Alexandrov K, Collins BM. Structural and thermodynamic 848 \nanalysis of the GFP:GFP-nanobody complex. Protein Sci. 2010;19(12):2389-401. doi: 849 \n10.1002/pro.519. PubMed PMID: 20945358; PMCID: PMC3009406. 850 \n62. Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, 851 \nAumonier S, Gotthard G, Royant A, Hink MA, Gadella TW, Jr. mScarlet: a bright 852 \nmonomeric red fluorescent protein for cellular imaging. Nat Methods. 2017;14(1):53-6. 853 \nEpub 20161121. doi: 10.1038/nmeth.4074. PubMed PMID: 27869816. 854 \n63. Hammond C, Braakman I, Helenius A. Role of N-linked oligosaccharide 855 \nrecognition, glucose trimming, and calnexin in glycoprotein folding and quality control. 856 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 34 \nProc Natl Acad Sci U S A. 1994;91(3):913-7. doi: 10.1073/pnas.91.3.913. PubMed 857 \nPMID: 8302866; PMCID: PMC521423. 858 \n64. Vassilakos A, Cohen-Doyle MF, Peterson PA, Jackson MR, Williams DB. The 859 \nmolecular chaperone calnexin facilitates folding and assembly of class I 860 \nhistocompatibility molecules. EMBO J. 1996;15(7):1495-506. PubMed PMID: 8612572; 861 \nPMCID: PMC450057. 862 \n65. Manganas LN, Trimmer JS. Calnexin regulates mammalian Kv1 channel 863 \ntrafficking. Biochem Biophys Res Commun. 2004;322(2):577-84. doi: 864 \n10.1016/j.bbrc.2004.06.182. PubMed PMID: 15325269. 865 \n66. Garty H, Palmer LG. Epithelial sodium channels: function, structure, and 866 \nregulation. Physiological Reviews. 1997. PubMed PMID: 9CD90C0A-7172-42BE-8B76-867 \n030D356A3C0B. 868 \n67. Jeong H, Clark S, Goehring A, Dehghani-Ghahnaviyeh S, Rasouli A, Tajkhorshid 869 \nE, Gouaux E. Structures of the TMC-1 complex illuminate mechanosensory 870 \ntransduction. Nature. 2022;610(7933):796-803. Epub 20221012. doi: 10.1038/s41586-871 \n022-05314-8. PubMed PMID: 36224384; PMCID: PMC9605866. 872 \n68. Goehring A, Lee C-H, Wang KH, Michel JC, Claxton DP, Baconguis I, Althoff T, 873 \nFischer S, Garcia KC, Gouaux E. Screening and large-scale expression of membrane 874 \nproteins in mammalian cells for structural studies. Nature Protocols. 2014;9(11):2574-875 \n85. doi: 10.1038/nprot.2014.173. PubMed PMID: 25299155; PMCID: PMC4291175. 876 \n69. Sorensen MV, Grossmann S, Roesinger M, Gresko N, Todkar AP, Barmettler G, 877 \nZiegler U, Odermatt A, Loffing-Cueni D, Loffing J. Rapid dephosphorylation of the renal 878 \nsodium chloride cotransporter in response to oral potassium intake in mice. Kidney Int. 879 \n2013;83(5):811-24. Epub 20130227. doi: 10.1038/ki.2013.14. PubMed PMID: 880 \n23447069. 881 \n70. Grimm PR, Taneja TK, Liu J, Coleman R, Chen YY, Delpire E, Wade JB, Welling 882 \nPA. SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-883 \nspecific manner. J Biol Chem. 2012;287(45):37673-90. Epub 20120912. doi: 884 \n10.1074/jbc.M112.402800. PubMed PMID: 22977235; PMCID: PMC3488044. 885 \n 886 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 35 \nFigure legends 887 \nFigure 1. Immunofluorescence staining of kidney tissue from homozygous 888 \nENaCγ-VF mice. 889 \na. Schematic of the tagged ENaCγ subunit produced from the ENaCγ-VF allele, which 890 \nincludes a C-terminal mVenus, 3C protease site, and 3xFLAG tag. b. Representative 891 \nimmunofluorescence image showing co-localization of mVenus and ENaCγ in kidney 892 \ntissue. mVenus signal was detected using a FITC-conjugated anti-GFP primary 893 \nantibody (goat, Rockland, 1:100) and Alexa Fluor 488 donkey anti-goat secondary 894 \nantibody. Endogenous ENaCγ was detected using a rabbit anti-ENaCγ primary antibody 895 \n(StressMarq, 1:500) targeting the C-terminus, and Alexa Fluor 647 donkey anti-rabbit 896 \nsecondary antibody. DAPI is 4’,6-diamidino-2-phenylindole. c. Close-up view 897 \nhighlighting localization of tagged ENaCγ.  898 \nFigure 2. The ENaCγ-VF mouse line is physiologically indistinguishable from 899 \nwild-type at baseline. 900 \na. Table showing plasma sodium (Na+) and potassium (K⁺) concentrations in wild-type 901 \nC57BL/6 and homozygous ENaCγ-VF mice under baseline conditions. b. Amiloride 902 \nresponse assay in wild-type C57BL/6 and ENaCγ-VF mice. The graph shows individual 903 \npaired responses to vehicle and amiloride for each mouse, with overlaid bars indicating 904 \nmean ± SEM (n = 6 per group). A two-way ANOVA revealed a significant effect of 905 \namiloride treatment compared with vehicle (***p < 0.0001), but no significant difference 906 \nbetween genotypes (p = 0.4533), indicating that the tagged allele supports normal 907 \nENaC function. c. Saturation binding of [³H]benzamil to kidney membranes. Increasing 908 \nconcentrations of [³H]benzamil (0.5-300 nM) were incubated with kidney membranes for 909 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 36 \n2 hours min at 22 °C. Specific binding (circles) was determined by subtracting 910 \nnonspecific binding measured in the presence of 100 µM unlabeled phenamil. Data 911 \nrepresent one experiment performed in triplicate; points show mean ± SD of triplicate 912 \nmeasurements. The curve was fit with a one-site specific binding model using nonlinear 913 \nregression (GraphPad Prism) to obtain Kd. 914 \nFigure 3. The ENaCγ-VF mouse enables subunit-specific detection of native ENaC 915 \ncomplexes using FSEC and SiMPull. 916 \na. Schematic workflow illustrating the experimental pipeline. Tissues (lung, kidney, 917 \ncolon) were harvested from ENaCγ-VF mice, homogenized in TRIS-based buffer, and 918 \nfollowed by solubilization. Cleared supernatants were injected onto a Superose 6 919 \nIncrease column for FSEC analysis, and fractions corresponding to high-molecular-920 \nweight complexes were subsequently analyzed by SiMPull. b. Representative FSEC 921 \ntraces showing mVenus fluorescence from lung, kidney, and colon lysates. Fluorescent 922 \npeaks indicate the presence of γ-containing ENaC complexes in each tissue. c. 923 \nRepresentative TIRF images of SiMPull experiments from lung lysates. mVenus-tagged 924 \nENaC complexes were captured using a biotinylated anti-GFP/mVenus nanobody. A 925 \nbackground control image was collected from a flow chamber lacking the nanobody to 926 \ndemonstrate specificity. d. Quantification of SiMPull signal from lung, kidney, and colon 927 \ntissues. Each condition represents data pooled from 40 TIRF images per sample, 928 \nanalyzed using the ComDet plugin in ImageJ. Statistical analysis was performed in 929 \nGraphPad Prism. 930 \nFigure 4. Dual-subunit detection of native ENaC complexes using 7B1-mScarlet 931 \nand the ENaCγ-VF mouse line. 932 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 37 \na. Cryo-EM map of human ENaC in complex with two Fabs, 10D4 and 7B1, shown in 933 \nside and top-down views. In the top-down view, a red box highlights the interaction site 934 \nbetween 7B1 and the α subunit. b. Sequence alignment of the experimentally 935 \ndetermined 7B1 epitope region in human and mouse ENaCα subunits, revealing high 936 \nconservation and supporting the potential for cross-reactivity with mouse ENaC. c. 937 \nSchematic representation of the 7B1-mScarlet Fab construct used to detect the ENaC α 938 \nsubunit. The variable regions of the 7B1 heavy and light chains were expressed as a 939 \nFab fragment in HEK293 cells, with mScarlet fused to the C-terminus of the heavy 940 \nchain. d. FSEC trace monitoring mVenus fluorescence from lung lysates of ENaCγ-VF 941 \nmice, showing the elution profile of γ-containing ENaC complexes. Dotted lines indicate 942 \nthe elution range for mVenus-tagged complexes (11-16 mL). e. FSEC trace from the 943 \nsame sample following 7B1-mScarlet fluorescence, revealing the elution profile of α-944 \ncontaining ENaC complexes. Dashed lines show that the α-associated signal is 945 \nrestricted to a narrower elution range (11-13 mL), suggesting that α-γ co-assemblies 946 \nrepresent a more defined subset of the total γ-containing complexes. f. Overlay of the 947 \nFSEC traces shown in panels d and e, plotted on the same axes to facilitate direct 948 \ncomparison of elution profiles and peak positions. The inset shows the full normalized 949 \ntraces for each condition. The main panel displays a magnified view of the gray-shaded 950 \nregion indicated in the inset, highlighting the relative alignment of the major peaks. 951 \nFigure 5. ENaC complex purification enables tissue-specific interactome analysis 952 \nby mass spectrometry. 953 \na. Schematic workflow illustrating the purification pipeline. Tissues (lung, kidney, colon) 954 \nfrom ENaCγ-VF mice were solubilized under non-denaturing conditions. γ-containing 955 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 38 \nENaC complexes were enriched by affinity capture via the C-terminal FLAG tag, eluted 956 \nby 3C protease cleavage, and analyzed by mass spectrometry. b. Representative FSEC 957 \ntraces from lung, kidney, and colon showing mVenus fluorescence, confirming 958 \nsuccessful enrichment of γ-containing complexes. The gray shaded region indicates the 959 \nfractions that were collected, pooled, and used for mass spectrometry analysis. A 960 \nsecond prominent peak is observed and corresponds to background fluorescence 961 \ndetected in kidney tissue, independent of ENaC-containing complexes. c. Mass 962 \nspectrometry results identifying proteins that co-purify with ENaCγ from each tissue. 963 \nData reveal both shared and tissue-enriched interactors, highlighting the power of this 964 \napproach to define native ENaC-associated complexes in a tissue-specific context. 965 \nFigure S1. Immunofluorescence staining of kidney tissue from heterozygous 966 \nENaCγ-VF mice. 967 \na. Representative immunofluorescence image showing co-localization of mVenus, 968 \nENaCγ, and aquaporin-2 in kidney tissue. mVenus signal was detected using a FITC-969 \nconjugated anti-GFP primary antibody (goat, Rockland, 1:100) and Alexa Fluor 488 970 \ndonkey anti-goat secondary antibody. Endogenous ENaCγ was detected using a rabbit 971 \nanti-ENaCγ primary antibody (StressMarq, 1:500) targeting the C-terminus, and Alexa 972 \nFluor 647 donkey anti-rabbit secondary antibody. Aquaporin-2 (AQP2) was stained 973 \nusing a mouse anti-AQP2 primary antibody (Santa Cruz, 1:50) and Alexa Fluor 555 974 \ndonkey anti-mouse secondary antibody. b. Representative immunofluorescence image 975 \nshowing co-localization of mVenus, ENaCα, and pNCC in kidney tissue. mVenus signal 976 \nwas detected using a FITC-conjugated anti-GFP primary antibody (goat, Rockland, 977 \n1:100) and Alexa Fluor 488 donkey anti-goat secondary antibody. Endogenous ENaCα 978 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 39 \nwas detected using a rabbit anti-ENaCα primary antibody (generous gift of J. Loffing69) 979 \ntargeting the C-terminus, and Alexa Fluor 647 donkey anti-rabbit secondary antibody. 980 \nThe phosphorylated form of the Na⁺-Cl⁻ cotransporter (NCC) was stained using a 981 \nchicken anti-NCC (generously provided by J. Wade70) and Cy3 donkey anti-chicken 982 \nsecondary antibody. c. Close-up view highlighting localization of tagged mVenus, 983 \nENaCα, and pNCC. * indicate DCT1 (pNCC+/ENaCα-); + indicate DCT2 984 \n(pNCC+/ENaCα+). 985 \nFigure S2. Validation of 7B1 -mScarlet as a tool for detecting mouse ENaC α. 986 \na. Schematic of the recombinant mouse ENaC subunit constructs used for validation. The 987 \nα and β subunits were expressed with wild -type sequences, while the γ subunit was C-988 \nterminally fused to mVenus, mirroring the configuration in ENaC γ-VF mice. b, c. FSEC 989 \ntraces showing mVenus (b) and mScarlet fluorescence (c) from lysates of cells expressing 990 \nrecombinant mouse ENaC. Co -elution of the two signals confirms that 7B1 -mScarlet 991 \nbinds specifically to complexes containing ENaC α, validating its use in detecting α 992 \nsubunits within γ-containing assemblies. d. Schematic illustration of ENaC complexes 993 \nlocated inside the cell and in the plasma membrane. Only surface ENaC is exposed to 994 \nmembrane-impermeable biotin. e. FSEC traces of purified surface -expressed ENaC 995 \nprotein (black trace) and intracellular ENaC pool (blue trace) monitored on the mScarlet 996 \nchannel fused to the 7B1 Fab. The difference in peak height is due to preexisting 997 \ndifferences in relative abundance and not a difference in 7B1 recognition. 998 \nFigure S3. Control purification workflow using wild-type mice to assess non-999 \nspecific binding. 1000 \nSchematic of the purification workflow applied to wild-type mouse tissues, performed in 1001 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\n \n 40 \nparallel with the ENaCγ-VF workflow. Although the protocol is identical, including tissue 1002 \nsolubilization, anti-FLAG affinity capture, and protease elution, wild-type mice lack the 1003 \nC-terminal 3xFLAG tag on ENaCγ and therefore cannot be captured by the anti-FLAG 1004 \nresin. This control serves to identify proteins that may non-specifically bind to the affinity 1005 \nresin and distinguishes true ENaC-associated interactors from background 1006 \ncontaminants in the ENaCγ-VF purification experiments. 1007 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\nFigure 1\na\nENaC γ mVenus\n3C protease \nsite 3xFLAG\nENaCγ-VF\nb\nmV enus ENaCγ\nDAPI Merged\nc\nmV enus ENaCγ\nDAPI Merged\n500 μm\n50 μm\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\nFigure 2\na\nENaCγ-VF \n(N=3)\nmmol/L\n147 (2.65)\n3.80 (0.520)\nWT (N=3)\nmmol/L\n146 (1.73)\n3.73 (0.351)\nNa+ Mean (SD)\nK+ Mean (SD)\nb\nc\nUrine Na+:K+ ratio (mmol/mmol)\n0\n5\n10\n15\nWT ENaCγ-VF\nVehicle\nAmiloride*** ***\nNS\n0 100 200 300\n0\n500\n1000\n1500\n2000\nnM Radioligand\nSpecific binding (cpm)\nENaCγ-VF\nWT\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\nFigure 3\nPEG\nGlass coverslip\nBiotin\nmVenus\nStreptavidin\nBiotinylated GFP \nnanobody ENaCγ\na\nENaCγ-VF\nSEC column\nFluorometer Elution volume (mL)\nProtein complex size\nLarge Small\nVoid\nFluorescence (a.u.)\nmVenus fluorescence\nb\nBackgroundmVenus\nc\nlungs kidneys colon\nd\nmVenus\nBackground\nmVenus\nBackground\nmVenus\nBackground\n0\n500\n1000\n1500Number of molecules\n****\n****\n****p<0.0001\n****\n12.5 15\nElution Volume (ml)\nColonColon\nKidneysKidneys\nLungsLungs\n 3xFLAG\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\nFigure 4\n7B1 light chain\n7B1 heavy chain\nmScarlet\nc\nd\n0 10 20 30\nElution Volume (mL)\nmVenus\nmVenus fluorescence\nmScarlet fluorescence\nNormalized\nmVenus\ne f\n0 10 20 30\nElution Volume (mL)\nmScarlet\nmScarletExcess \n7B1-\nmScarlet\na\nOut γ\nβ\nα\nγ\nβ α\n7B1\nFab\n10D4\nFab\n10D4\nFab\n7B1\nFab\nIn\n90°\nb\nhENaCα LPETLPSLE\nmENaCα LPDTSPALE\n263 271\n290 298\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint \n\nFigure 5\n10 15 20\nENaCγ-VF\nlungs\n ENaCγ-VF tissue\n Supernatant\n Affinity isolation\n FSEC purification\n Mass spectrometry\n Solubilize tissue\n Anti-FLAG resin\n 3xFLAG\n 3C protease \ncleavage\n Concentrate \neluted fractions\nmVenus fluorescence\nElution volume (mL)\n10 15 20\nENaCγ-VF\nkidneys\nmVenus fluorescence\nElution volume (mL)\n10 15 20\nENaCγ-VF\ncolon\nmVenus fluorescence\nElution volume (mL)\nba\nc\nWT ENaCγ-VF \n0\n0\n0\n0\n0\n0\n8\n3\n3\n4\n2\n0\nKidneyKidney Lungs Colon\nSCNNG_MOUSE 33\nSCNNB_MOUSE 25\nSCNNA_MOUSE 14\nDHI2_MOUSE 19\nCALX_MOUSE 14\nNED4L_MOUSE 11\n112\n136\n52\n46\n0\n14\n Pool fractions\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701393doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}