Transmissible Gastroenteritis Virus Nucleocapsid Protein Interacts with Na+/H+ Exchanger 3 To Reduce Na+/H+ Exchanger Activity and Promote Piglet Diarrhea.

Transmissible gastroenteritis virus (TGEV) is member of the family Coronaviridae and mainly causes acute diarrhea. TGEV infection is characterized by vomiting, watery diarrhea, and severe dehydration, resulting in high mortality rates in neonatal piglets. TGEV infection symptoms are related to an imbalance of sodium absorption in small intestinal epithelial cells; however, the etiology of sodium imbalance diarrhea caused by TGEV remains unclear. In this study, we performed transcriptomic analysis of intestinal tissues from infected and healthy piglets and observed that the expression of NHE3, encoding Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, was significantly reduced upon TGEV infection. We also showed that specific inhibition of intestinal NHE3 activity could lead to the development of diarrhea in piglets. Furthermore, we revealed an interaction between TGEV N protein and NHE3 near the nucleus. The binding of TGEV N to NHE3 directly affected the expression and activity of NHE3 on the cell surface and affected cellular electrolyte absorption, leading to diarrhea. Molecular docking and computer-aided screening techniques were used to screen for the blocker of the interaction between TGEV N and NHE3, which identified irinotecan. We then demonstrated that irinotecan was effective in relieving TGEV-induced diarrhea in piglets. These findings provide new insights into the mechanism of TGEV-induced sodium imbalance diarrhea and could lead to the design of novel antiviral strategies against TGEV. IMPORTANCE A variety of coronaviruses have been found to cause severe diarrhea in hosts, including TGEV; however, the pathogenic mechanism is not clear. Therefore, prompt determination of the mechanism and identification of efficient therapeutic agents are required, both for public health reasons and for economic development. In this study, we demonstrated that NHE3 is the major expressed protein of NHEs in the intestine, and its expression decreased by nearly 70% after TGEV infection. Also, specific inhibition of intestinal NHE3 resulted in severe diarrhea in piglets. This demonstrated that NHE3 plays an important role in TGEV-induced diarrhea. In addition, we found that TGEV N directly regulates NHE3 expression and activity through protein-protein interaction, which is essential to promote diarrhea. Molecular docking and other techniques demonstrated that irinotecan could block the interaction and diarrhea caused by TGEV. Thus, our results provide a basis for the development of novel therapeutic agents against TGEV and guidance for the development of drugs for other diarrhea-causing coronaviruses.

Functional Assessment In Vivo of the Mouse Homolog of the Human Ala-9-Ser NHE6 Variant.

Christianson syndrome (CS) is an X-linked neurogenetic disorder resulting from loss-of-function (LoF) mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). NHE6 regulates proton efflux from endosomes and, thus, participates in regulating cargo processing and trafficking. LoF mutations in NHE6 cause aberrant acidification of endosomes. While CS arises in males generally due to clear LoF mutations, other potentially hypomorphic variants have emerged, yet most of these variants have not been evaluated for functional effects, particularly in vivo Here we characterize an SLC9A6 variant that has been previously reported in patients, yet now also appears in exome datasets of largely control individuals-c.25G>T, p.A9S. By heterologous expression in cell lines, we show that human NHE6A9S is expressed and localizes in a manner comparable to control NHE6. By genome editing, we generated the equivalent NHE6 mutation in mouse-p.A11S-and determined that male NHE6A11S mice have normal brain size at 6 months of age and do not show cerebellar degeneration or defective neuronal arborization. Neurons from male NHE6A11S mice also did not demonstrate an abnormality in intraendosomal pH compared with controls. These findings are in contrast to findings in NHE6-null mice previously reported and indicate that the NHE6A11S variant functions at a level equivalent to control NHE6 for many of the assays performed. These data stand in support of the population genetic data, which are also evaluated here, indicating that the A9S variant is unlikely to confer disease susceptibility with high penetrance.

Simulating the function of sodium/proton antiporters.

The molecular basis of the function of transporters is a problem of significant importance, and the emerging structural information has not yet been converted to a full understanding of the corresponding function. This work explores the molecular origin of the function of the bacterial Na+/H+ antiporter NhaA by evaluating the energetics of the Na+ and H+ movement and then using the resulting landscape in Monte Carlo simulations that examine two transport models and explore which model can reproduce the relevant experimental results. The simulations reproduce the observed transport features by a relatively simple model that relates the protein structure to its transporting function. Focusing on the two key aspartic acid residues of NhaA, D163 and D164, shows that the fully charged state acts as an Na+ trap and that the fully protonated one poses an energetic barrier that blocks the transport of Na+. By alternating between the former and latter states, mediated by the partially protonated protein, protons, and Na+ can be exchanged across the membrane at 2:1 stoichiometry. Our study provides a numerical validation of the need of large conformational changes for effective transport. Furthermore, we also yield a reasonable explanation for the observation that some mammalian transporters have 1:1 stoichiometry. The present coarse-grained model can provide a general way for exploring the function of transporters on a molecular level.

Ocean warming and acidification modulate energy budget and gill ion regulatory mechanisms in Atlantic cod (Gadus morhua).

Ocean warming and acidification are threatening marine ecosystems. In marine animals, acidification is thought to enhance ion regulatory costs and thereby baseline energy demand, while elevated temperature also increases baseline metabolic rate. Here we investigated standard metabolic rates (SMR) and plasma parameters of Atlantic cod (Gadus morhua) after 3-4 weeks of exposure to ambient and future PCO2 levels (550, 1200 and 2200 µatm) and at two temperatures (10, 18 °C). In vivo branchial ion regulatory costs were studied in isolated, perfused gill preparations. Animals reared at 18 °C responded to increasing CO2 by elevating SMR, in contrast to specimens at 10 °C. Isolated gills at 10 °C and elevated PCO2 (≥1200 µatm) displayed increased soft tissue mass, in parallel to increased gill oxygen demand, indicating an increased fraction of gill in whole animal energy budget. Altered gill size was not found at 18 °C, where a shift in the use of ion regulation mechanisms occurred towards enhanced Na(+)/H(+)-exchange and HCO3 (-) transport at high PCO2 (2200 µatm), paralleled by higher Na(+)/K(+)-ATPase activities. This shift did not affect total gill energy consumption leaving whole animal energy budget unaffected. Higher Na(+)/K(+)-ATPase activities in the warmth might have compensated for enhanced branchial permeability and led to reduced plasma Na(+) and/or Cl(-) concentrations and slightly lowered osmolalities seen at 18 °C and 550 or 2200 µatm PCO2 in vivo. Overall, the gill as a key ion regulation organ seems to be highly effective in supporting the resilience of cod to effects of ocean warming and acidification.

Concurrent speed endurance and resistance training improves performance, running economy, and muscle NHE1 in moderately trained runners.

The purpose of this study was to examine whether speed endurance training (SET, repeated 30-s sprints) and heavy resistance training (HRT, 80-90% of 1 repetition maximum) performed in succession are compatible and lead to performance improvements in moderately trained endurance runners. For an 8-wk intervention period (INT) 23 male runners [maximum oxygen uptake (V̇O(2max)) 59 ± 1 ml·min(-1)·kg(-1); values are means ± SE] either maintained their training (CON, n = 11) or performed high-intensity concurrent training (HICT, n = 12) consisting of two weekly sessions of SET followed by HRT and two weekly sessions of aerobic training with an average reduction in running distance of 42%. After 4 wk of HICT, performance was improved (P < 0.05) in a 10-km run (42:30 ± 1:07 vs. 44:11 ± 1:08 min:s) with no further improvement during the last 4 wk. Performance in a 1,500-m run (5:10 ± 0:05 vs. 5:27 ± 0:08 min:s) and in the Yo-Yo IR2 test (706 ± 97 vs. 491 ± 65 m) improved (P < 0.001) only following 8 wk of INT. In HICT, running economy (189 ± 4 vs. 195 ± 4 ml·kg(-1)·km(-1)), muscle content of NHE1 (35%) and dynamic muscle strength was augmented (P < 0.01) after compared with before INT, whereas V̇O(2max), muscle morphology, capillarization, content of muscle Na(+)/K(+) pump subunits, and MCT4 were unaltered. No changes were observed in CON. The present study demonstrates that SET and HRT, when performed in succession, lead to improvements in both short- and long-term running performance together with improved running economy as well as increased dynamic muscle strength and capacity for muscular H(+) transport in moderately trained endurance runners.

Assessment of myocardial viability by intracellular 23Na magnetic resonance imaging.

BACKGROUND: Because of rapid changes in myocardial intracellular Na+ (Na+(i)) during ischemia and reperfusion (R), 23Na magnetic resonance imaging (MRI) appears to be an ideal diagnostic modality for early detection of myocardial ischemia and viability. So far, cardiac 23Na MRI data are limited and mostly concerned with imaging of total Na+. For proper interpretation, imaging of both Na+(i) and extracellular Na+ is essential. In this study, we tested whether Na+(i) imaging can be used to assess viability after low-flow (LF) ischemia. METHODS AND RESULTS: Isolated rat hearts were subjected to LF (1%, 2%, or 3% of control coronary flow) and R. A shift reagent was used to separate Na+(i) and extracellular Na+ resonances. Acquisition-weighted 23Na chemical shift imaging (CSI) was alternated with 23Na MR spectroscopy. Already during control perfusion, Na+(i) could be clearly seen on the images. Na+(i) image intensity increased with increasing severity of ischemia. During R, Na+(i) image intensity remained highest in 1% LF hearts. Not only did we find very good correlations between Na+(i) image intensity at end-R and end-diastolic pressure (R=0.85, P<0.001) and recovery of the rate-pressure product (R=-0.88, P<0.001) at end-R, but most interestingly, also Na+(i) image intensity at end-LF was well correlated with end-diastolic pressure (R=0.78, P<0.01) and with recovery of the rate-pressure product (R=-0.81, P<0.01) at end-R. Furthermore, Na+(i) image intensity at end-LF was well correlated with creatine kinase release during R (R=0.79, P<0.05) as well as with infarct size (R=0.77, P<0.05). CONCLUSIONS: These data indicate that 23Na CSI is a promising tool for the assessment of myocardial viability.

ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids.

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.

Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.