ells expressing ACE2 are potential targets of SARS-CoV-2 infection 1,2. Studies based on single-cell RNA sequencing (scRNA-seq) of lung cells have identified type II pneumocytes, ciliated cells and transient secretory cells as the main types of ACE2-expressing cell 3,4. Furthermore, ACE2 was proposed to be an ISG, on the basis of its inducible expression in cells treated with interferons (IFNs) or infected by viruses that induce IFN responses, such as influenza 4,5. These findings implied that the induction of ACE2 expression in IFN-high conditions could result in an amplified risk of SARS-CoV-2 infection 4,5. Concerns could also be raised about possible ACE2-inducing side effects of IFN-based treatments proposed for COVID-19 (refs. 6-9). ACE2 plays multiple roles in normal physiological conditions and as part of the host tissue-protective machinery in damaging conditions, including viral infections. As a terminal carboxypeptidase, ACE2 cleaves a single carboxy-terminal residue from peptide hormones such as angiotensin II and des-Arg9-bradykinin. ACE and ACE2 belong to the renin-angiotensin-aldosterone system, which regulates blood pressure and fluid-electrolyte balance; dysfunction of this system contributes to comorbidities in COVID-19 (refs. 10,11). des-Arg9-bradykinin is generated from bradykinin and belongs to the kallikrein-kinin system, which is critical in regulating vascular leakage and pulmonary edema, early signs of severe COVID-19 (refs. 12,13). High plasma angiotensin II levels were found to be responsible for coronavirus-associated acute respiratory distress syndrome (ARDS), lung damage and high mortality in mouse models 14,15 and as a predictor of lethality in avian influenza in humans 16,17. In the same conditions, ACE2, which decreases the levels of angiotensin II, was identified as a protective factor. The hijacking of the normal host tissue-protective machinery guarded by ACE2 was suggested as a mechanism through which SARS-CoV-2 could infect more cells 4,5. Thus, it is critically important to identify factors affecting ACE2 expression in normal physiological processes and during viral infections and associated pathologies, such as in COVID-19. Herein, aiming to explore the IFN-inducible expression of ACE2 and its role in SARS-CoV-2 infection, we identified a novel, truncated isoform of ACE2, which we designate as dACE2. We then showed that dACE2, but not ACE2, is induced in various human cell types by IFNs and viruses; this information is important to consider for future therapeutic strategies and understanding COVID-19 susceptibility and outcomes. Results dACE2 is a novel inducible isoform of ACE2. To address the extent to which IFNs induce the expression of ACE2 in human cells, we used our existing RNA-seq dataset (NCBI Sequence Read Archive (SRA): PRJNA512015) of a breast cancer cell line T47D infected with Sendai virus (SeV), known to be a strong inducer of IFNs and ISGs 18-20. IFNs were not expressed in T47D cells at baseline, but SeV strongly induced expression of IFNB1, a type I IFN, an...

The members of the cytoplasmic 70-kDa heat shock protein family are involved in appropriate folding and trafficking of newly synthesized proteins in the cell. Hsc70, which is expressed constitutively, and Hsp70, the expression of which is stress-and heat shock-induced, are often considered to have similar cellular functions in this regard, but there are suggestions that the intracellular functions of these homologous but not identical proteins may differ. We tested the hypothesis that Hsc70 and Hsp70 would have differential effects on the expression of the epithelial sodium channel (ENaC). In Xenopus oocytes, overexpression of human Hsc70 decreased the functional (defined as amiloride-sensitive whole-oocyte current) and surface expression of murine ENaC (mENaC) in a concentration-dependent fashion. In contrast, coinjection of a moderate amount of Hsp70 cRNA (10 ng) increased the functional and surface expression of mENaC, whereas a higher amount of coinjected Hsp70 cRNA (30 ng) decreased mENaC functional and surface expression. The increase in mENaC functional expression with coinjection of 10 ng of Hsp70 cRNA was antagonized by the additional coinjection of Hsc70 cRNA in a concentration-dependent fashion. These data are consistent with Hsc70 and Hsp70 having differential and antagonistic effects with regard to the intracellular trafficking of mENaC in oocytes, which may have an impact on our understanding and potential treatment of diseases of aberrant ion channel trafficking.chaperone ͉ Xenopus oocyte ͉ cystic fibrosis ͉ ENaC ͉ antagonism

A common human epithelial sodium channel (ENaC) polymorphism, ␣T663A, is present in the cytoplasmic C terminus of the ␣-subunit, although it is unclear whether this polymorphism segregates with blood pressure. We examined whether this polymorphism was associated with differences in functional Na ؉ channel expression. Whole cell amiloride-sensitive currents in Xenopus oocytes expressing wild type channels (␣T663␤␥) were significantly ϳ1.3-2.0-fold higher than currents measured in oocytes expressing channels with an Ala, Gly or Leu, or Lys at position ␣663. In contrast, differences in functional human ENaC expression were not observed with oocytes expressing channels having Thr (wild type), Ser, or Asp at this position. The surface expression of channels, measured using an epitopetagged ␤-subunit, was significantly reduced in oocytes expressing ␣T663A␤␥ when compared with oocytes expressing ␣T663␤␥. The corresponding polymorphism was generated in the mouse ␣-subunit (m␣A692T) and was not associated with differences in functional ␣␤␥-mouse ENaC expression. The polymorphism is present in a region that is not well conserved between human and mouse. We generated a mouse/human chimera by replacement of the distal C terminus of the mouse ␣-subunit with the distal C terminus of the human ␣-subunit. Co-expression of this m(1-678)/h(650 -669)T663A chimera with mouse ␤␥ led to a significant reduction in whole cell Na ؉ currents and surface expression when compared with m(1-678)/h(650 -669)T663-m␤␥. Our results suggest that h␣T663A is a functional polymorphism that affects human ENaC surface expression.Epithelial sodium channels (ENaC) 1 are expressed in principal cells in the late distal convoluted tubule and collecting tubule, where they serve as a final site for reabsorption of Na ϩ from the glomerular ultrafiltrate. Volume regulatory hormones, such as aldosterone, have a key role in modifying rates of renal tubular Na ϩ reabsorption through regulation of functional ENaC expression at the apical plasma membrane (1). Epithelial Na ϩ channels are composed of three structurally related subunits, termed ␣-, ␤-, and ␥-ENaC that likely assemble as a ␣2,␤1,␥1 tetramer (2, 3), although an alternative subunit stoichiometry has been proposed (4). The three subunits share limited (ϳ30 -40%) sequence identity but share a common topology of two membrane-spanning domains and intracellular N and C termini (5-7).Changes in ENaC functional expression are associated with alterations in blood pressure (8, 9). Na ϩ channel gain-of-function mutations have been identified in patients with Liddle's syndrome, a disorder characterized by volume expansion, hypokalemia, and hypertension (10, 11). ENaC loss-of-function mutations have been identified in patients with type I pseudohypoaldosteronism, a disorder characterized by volume depletion, hypotension, and hyperkalemia (12, 13). Some common human ENaC polymorphisms may segregate with blood pressure (i.e. ␤T594M) (14), suggesting that ENaC polymorphisms that alter functional channel expression m...