Abstract
Fluxes of monovalent ions through the multiple pathways of the plasma membrane are highly interdependent, and their assessment by direct measurement is difficult or even impossible. Computation of the entire flux balance helps to identify partial flows and study the functional expression of individual transporters. Our previous computation of unidirectional fluxes in real cells ignored the ubiquitous cotransporters NKCC and KCC. Here, we present an analysis of the entire balance of unidirectional Na+, K+, and Cl– fluxes through the plasma membrane in human lymphoid U937 cells, taking into account not only the Na/K pump and electroconductive channels but all major types of cotransporters NC, NKCC, and KCC. Our calculations use flux equations based on the fundamental principles of macroscopic electroneutrality of the system, water balance, and the generally accepted thermodynamic dependence of ion fluxes on the driving force, and they do not depend on hypotheses about the molecular structure of the channel and transporters. A complete list of the major inward and outward Na+, K+, and Cl– fluxes is obtained for human lymphoid U937 cells at rest and during changes in the ion and water balance for the first 4 h of staurosporine-induced apoptosis. It is shown how the problem of the inevitable multiplicity of solutions to the flux equations, which arises with an increase in the number of ion pathways, can be solved in real cases by analyzing the ratio of ouabain-sensitive and ouabain-resistant parts of K+ (Rb+) influx (OSOR) and using additional experimental data on the effects of specific inhibitors. It is found that dynamics of changes in the membrane channels and transporters underlying apoptotic changes in the content of ions and water in cells, calculated without taking into account the KCC and NKCC cotransporters, differs only in details from that calculated for cells with KCC and NKCC. The developed approach to the assessment of unidirectional fluxes may be useful for understanding functional expression of ion channels and transporters in other cells under various conditions. Attached software allows reproduction of all calculated data under presented conditions and to study the effects of the condition variation.
Highlights
Apoptosis is one of the three main types of cell death, along with autophagy and necrosis itself (Galluzzi et al, 2018)
A hallmark of apoptosis is a specific cell shrinkage or, at least, the absence of swelling and rupture of the plasma membrane (Yurinskaya et al, 2005). This is due to specific apoptotic changes in monovalent ion homeostasis, which is closely related to cell water balance regulation (Maeno et al, 2000, 2012; Okada et al, 2001; Lang et al, 2005; Lang and Hoffmann, 2012, 2013)
The quantitative study of changes in channels and transporters responsible for specific apoptotic changes in the balance of Na+, K+, and Cl− has been based on the use of staurosporine-treated U937 cells and computer modeling without considering KCC and NKCC cotransporters (Yurinskaya et al, 2019)
Summary
Apoptosis is one of the three main types of cell death, along with autophagy and necrosis itself (Galluzzi et al, 2018). The quantitative study of changes in channels and transporters responsible for specific apoptotic changes in the balance of Na+, K+, and Cl− has been based on the use of staurosporine-treated U937 cells and computer modeling without considering KCC and NKCC cotransporters (Yurinskaya et al, 2019) These cation-coupled Cl− cotransporters of the gene family SLC12 attract much attention in the recent decade (Gagnon and Delpire, 2013; Jentsch, 2016; Delpire and Gagnon, 2018). Progress in the molecular biology of the cation-coupled Cl− cotransporters is exciting; their functional expression and role in maintaining Cl− homeostasis in non-polarized cells is investigated much worse because electrophysiological methods cannot be applied here, and possible tools are rather limited It was said: “Electrical activity in neurons requires a seamless functional coupling between plasmalemmal ion channels and ion transporters. Ion channels have been studied intensively for several decades, research on ion transporters is in its infancy” (Kaila et al, 2014)
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