The 370 Da inhibitor of the sodium pump in the plasma of haemodialysis patients

Background: Previous studies have shown that a molecule of mass 370 Da that inhibits the sodium pump can be extracted from human placentas and from the concentrated plasma or ultrafiltrate of volume-expanded patients. Aim: To study the abundance of the 370 Da molecule and its changes across dialysis in a population of patients with renal failure treated by haemodialysis. Methods: 4 mL pre- and post-dialysis blood samples (2 mL plasma) were taken from patients receiving intermittent haemodialysis and analysed by high performance liquid chromatography (HPLC) coupled to high sensitivity mass spectrometry. Results: In over half the study population, the 370 Da molecule was present in an abundance that exceeded the limit of quantitation. Most patients experienced a marked fall in the abundance of the molecule over a haemodiafiltration (HDF) session, though exceptions were seen in two individuals both of whom showed clear evidence for the presence of two structural isomers of the 370 Da molecule. Conclusions: Advanced renal failure is frequently accompanied by an increased abundance of a 370 Da inhibitor of the sodium pump and that abundance is strongly impacted by haemodialysis. The technique described here could readily be applied to other clinical situations where sodium pump inhibition might be anticipated, such as hypertension, pregnancy and fetal medicine and thereby lead to a better understanding of the physiology and patho-physiology of these conditions.

Cation-Chloride Cotransporters, Na/K Pump, and Channels in Cell Water and Ion Regulation: In silico and Experimental Studies of the U937 Cells Under Stopping the Pump and During Regulatory Volume Decrease

Cation-coupled chloride cotransporters play a key role in generating the Cl– electrochemical gradient on the cell membrane, which is important for regulation of many cellular processes. However, a quantitative analysis of the interplay between numerous membrane transporters and channels in maintaining cell ionic homeostasis is still undeveloped. Here, we demonstrate a recently developed approach on how to predict cell ionic homeostasis dynamics when stopping the sodium pump in human lymphoid cells U937. The results demonstrate the reliability of the approach and provide the first quantitative description of unidirectional monovalent ion fluxes through the plasma membrane of an animal cell, considering all the main types of cation-coupled chloride cotransporters operating in a system with the sodium pump and electroconductive K+, Na+, and Cl– channels. The same approach was used to study ionic and water balance changes associated with regulatory volume decrease (RVD), a well-known cellular response underlying the adaptation of animal cells to a hypoosmolar environment. A computational analysis of cell as an electrochemical system demonstrates that RVD may happen without any changes in the properties of membrane transporters and channels due to time-dependent changes in electrochemical ion gradients. The proposed approach is applicable when studying truly active regulatory processes mediated by the intracellular signaling network. The developed software can be useful for calculation of the balance of the unidirectional fluxes of monovalent ions across the cell membrane of various cells under various conditions.

Elevated Sodium Pump α3 Subunit Expression Promotes Colorectal Liver Metastasis via the p53-PTEN/IGFBP3-AKT-mTOR Axis

The sodium pump α3 subunit is associated with colorectal liver metastasis. However, the underlying mechanism involved in this effect is not yet known. In this study, we found that the expression levels of the sodium pump α3 subunit were positively associated with metastasis in colorectal cancer (CRC). Knockdown of the α3 subunit or inhibition of the sodium pump could significantly inhibit the migration of colorectal cancer cells, whereas overexpression of the α3 subunit promoted colorectal cancer cell migration. Mechanistically, the α3 subunit decreased p53 expression, which subsequently downregulated PTEN/IGFBP3 and activated mTOR, leading to the promotion of colorectal cancer cell metastasis. Reciprocally, knockdown of the α3 subunit or inhibition of the sodium pump dramatically blocked this effect in vitro and in vivo via the downregulation of mTOR activity. Furthermore, a positive correlation between α3 subunit expression and mTOR activity was observed in an aggressive CRC subtype. Conclusions: Elevated expression of the sodium pump α3 subunit promotes CRC liver metastasis via the PTEN/IGFBP3-mediated mTOR pathway, suggesting that sodium pump α3 could represent a critical prognostic marker and/or therapeutic target for this disease.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-Pi form of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The two pumps show significant differences in structure in and around Site I, but which are critical for blocking K+ binding in the gastric pump and contribute to binding in the sodium pump is unclear. We have a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I based on the structure of the sodium pump. The number of bound Rb+, determined by its anomalous dispersion, remains one in the luminal-open E2BeF form of the Lys791Ser single mutant and Lys791Ser/Glu820Asp double mutant, mutation that could create space and may directly bind the cation. We next introduced mutations in peripheral residues Try340Asn and Glu936Val. A strong and spread-out Rb+ anomalous density observed in the quadruple mutant suggests that a certain population ATPases has two Rb+ bound. We then added gate-closing mutation Try799Trp and determined its cryo-EM structure in the occluded E2-AlF form. This quintuple mutant unambiguously has two separate densities at the cation-binding site. The step-wise construction of the K+ binding site offers new insight into how it is blocked in the one pump and constituted in the other.

Sodium Pump Na+/K+ ATPase Subunit α1 Targeted Positron Emission Tomography Imaging of Hepatocellular Carcinoma in Mouse Models

Purpose: Positron emission tomography (PET) imaging was not efficiently used in early diagnosis of hepatocellular carcinoma (HCC) due to lack of appropriate tracers. Sodium pump Na+/K+ ATPase subunit α1 (NKAα1) emerges to be a prognostic and diagnostic biomarker of HCC. Here we investigated the feasibility of 18F-ALF-NOTA-S3, a PET tracer based on an NKAα1 peptide, to detect small HCC. Methods: GEPIA database was searched to obtain the expression characteristics of NKAα1 in HCC and its relationship with the prognosis. PET/CT was performed in orthotopic, diethylnitrosamine (DEN)-induced and genetically engineered HCC mouse models to evaluate the use of 18F-ALF-NOTA-S3 to detect HCC lesions. Results: NKAα1 is overexpressed in early HCC with a high positive rate and correlates with poor survival. In orthotopic, DEN-induced and genetically engineered HCC mouse models, PET/CT imaging showed high accumulation of 18F-ALF-NOTA-S3 in the tumor. The tumor-to-liver ratios are 2.56 ± 1.02, 4.41 ± 1.09 and 4.59 ± 0.65 respectively. Upregulated NKAα1 expression in tumors were verified by immunohistochemistry. Furthermore, 18F-ALF-NOTA-S3 has the ability to detect small HCC lesions with diameters of 2-5mm. Conclusion: NKAα1 may serve as a suitable diagnostic and prognostic biomarker for HCC. 18F-ALF-NOTA-S3 shows great potential for PET imaging of HCC.