Background: Epithelial sodium channel (ENaC)  and ␥ subunits are modified by Cys palmitoylation. Results: Palmitoylation of the ␥ subunit activates ENaCs by increasing channel open probability. Conclusion: ␥ subunit palmitoylation has a dominant role in activating ENaCs compared with  subunit palmitoylation. Significance: ENaC palmitoylation is an important post-translational mechanism of channel regulation.
Phosphorylation of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular potassium concentration. High extracellular [K+] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K+] fails to dephosphorylate NCC, suggesting an interplay between the NCC-activating and inactivating switches. Here, we explored the role of SPAK and intracellular chloride concentration in the rapid effects of extracellular K+ on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in HEK cells and ex vivo in kidney slices by high [K+]. Acute high K+ challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of WNK bodies. In line with the postulate of interplay between activating and inactivating switches, we found that the ON switch, represented by WNK4-SPAK, must be turned off for rapid NCC dephosphorylation by high [K+]. Longer term WNK-SPAK mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K+ loading. Although blocking PP1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, were essential for NCC dephosphorylation. Overall our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases; with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K+.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters through the airways and infects the lungs, causing lethal pulmonary damage in vulnerable patients. This virus contains spike proteins on its envelope that binds to human angiotensin-converting enzyme 2 (hACE2) expressed on the surface of airway cells, enabling entry of the virus for causing infection1,2. In severe cases, the virus enters the circulatory system, contributing to multiorgan failure. Soluble form of hACE2 binds to SARS-CoV-2 spike protein and prevents viral entry into target cells3. Moreover, soluble recombinant ACE2 ameliorates lung injury4 but its short half-life limits its therapeutic utility5. Here, we engineered synthetic mRNA to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. Novel lipid nanoparticles (LNPs) were used to package mRNA and transfect mammalian cells for enhanced production of secreted proteins. Intravenously administered LNP led to hepatic delivery of the mRNA. This elicited secretion of hsACE2 into the blood circulation within 2 h, and levels of circulating hsACE2 peaked at 6 h and gradually decreased over several days. Since the primary site of entry and pathogenesis for SARS-CoV-2 is the lungs, we instilled LNPs into the lungs and were able to detect hsACE2 in the bronchoalveolar lavage fluid within 24 h and lasted for 48 h. Through co-immunoprecipitation, we found that mRNA-generated hsACE2 was able to bind with the receptor binding domain of the SARS-CoV-2 spike protein. Furthermore, hsACE2 was able to strongly inhibit (over 90%) SARS-CoV-2 pseudovirus infection. Our proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed for pulmonary and extrapulmonary neutralization of SARS-CoV-2 and open new treatment opportunities for COVID-19.
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) can cause lethal pulmonary damage in humans. It contains spike proteins on its envelope that bind to human angiotensin‐converting enzyme 2 (hACE2) expressed on airway cells, enabling entry of the virus, and causing infection. The soluble form of hACE2 binds SARS‐CoV‐2 spike protein, prevents viral entry into target cells, and ameliorates lung injury; however, its short half‐life limits therapeutic utilities. Here, synthetic mRNA is engineered to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. A novel lipid nanoparticle (LNP) is used for packaging and delivering mRNA to cells to produce hsACE2 proteins. Intravenously administered LNP delivers mRNA to hepatocytes, leading to the production of circulatory hsACE2 initiated within 2 h and sustained over several days. Inhaled LNP results in lung transfection and secretion of mucosal hsACE2 to lung epithelia, the primary site of entry and pathogenesis for SARS‐CoV‐2. Furthermore, mRNA‐generated hsACE2 binds to the receptor‐binding domain of the viral spike protein. Finally, hsACE2 effectively inhibits SARS‐CoV‐2 and its pseudoviruses from infecting host cells. The proof of principle study shows that mRNA‐based nanotherapeutics can be potentially deployed to neutralize SARS‐CoV‐2 and open new treatment opportunities for coronavirus disease 2019 (COVID‐19).
Edited by Henrik G. DohlmanThe epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (␣, , and ␥). We previously reported that cytoplasmic residues on the  (Cys-43 and Cys-557) and ␥ (␥Cys-33 and ␥Cys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, ␥ subunit palmitoylation had a dominant role over  subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when ␥ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking  subunit palmitoylation sites.  subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking  and ␥ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCD cl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCD cl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na ؉ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.ENaCs 2 are amiloride-sensitive Na ϩ channels that are found in high resistance epithelia and other tissues. In the aldosterone-sensitive distal nephron (ASDN), ENaC-dependent Na ϩ transport has an important role in the maintenance of extracellular fluid volume and blood pressure, as well as extracellular K ϩ homeostasis. In the lung, ENaC has a role in regulating airway surface fluid volume, mucociliary clearance, and alveolar fluid volume (1-3). The channels are composed of three homologous subunits (termed ␣, , and ␥), each with two transmembrane domains, a large extracellular loop and cytoplasmic N and C termini (3-5).ENaCs are regulated by signaling pathways that modulate its membrane trafficking, residency on the plasma membrane, and degradation (6 -8). ENaCs are also regulated by a variety of extracellular factors that affect its open probability (P o ). These include extracellular cations (H ϩ , Na ϩ , and other metals), anions (Cl Ϫ ), laminar shear stress, and proteases that cleave ENaC subunits at specific sites and release embedded inhibitory tracts (1, 2, 9 -14). Intracellular phosphorylation, inositol phospholipids, and cytoplasmic Cys palmitoylation also regulate ENaC P o (15-21).Cys palmitoylation of soluble and transmembrane proteins is a reversible post-translational modification that increases protein surface hydr...
Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.
Hypomagnesemia is associated with reduced kidney function and life-threatening complications and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg2+ excretion and, via activity of the Na+-Cl− cotransporter (NCC), also plays a key role in K+ homeostasis by metering Na+ delivery to distal segments. Little is known about the mechanisms by which plasma Mg2+ concentration regulates NCC activity and how low-plasma Mg2+ concentration and K+ concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg2+ restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg2+ restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K+ restriction. Mg2+ restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg2+ or combined Mg2+-K+ restriction but were dramatically lower compared with a low-K+ diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na+ delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg2+-K+ restriction, but, surprisingly, NCC was not activated after long-term K+ restriction despite lower plasma K+ concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K+ restriction.
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