PMC

Dependence of reverse-mode NHE1 proton flux on extracellular pH with 70 and 10 mM of cytoplasmic Na, a cytoplasmic pH of 7.6 and nominally no extracellular Na_o (n = 4).

Dependence of forward-mode NHE1 proton flux on extracellular pH with 140 and 20 mM of extracellular Na with a cytoplasmic pH of 6.8 and with nominally 0 mM of cytoplasmic Na (n = 4).

Simulations of the cis–trans interactions of Na and H under near zero-trans conditions, as in . (A) The dependence of half-maximal extracellular Na concentration of NHE activity on cytoplasmic pH under the conditions shown in together with data points from . (B) The dependence of half-maximal cytoplasmic proton concentrations, based on fits of simulated data to Michaelis-Menton relations, on the extracellular Na concentration under the conditions of together with data points from .

Cytoplasmic Na dependence of NHE1 proton flux with 140 mM of extracellular Na, nominally no cytoplasmic Na, and with an extracellular pH of 8.2. Filled circles show average data from four individual experiments where pH_i was changed serially in the same cell via pipette perfusion (for details see Materials and methods). Open circles show data for 140 mM of extracellular Na obtained in the experiments presented in . The best Hill equation fit to all data points in the figure is given as a solid line.

Simulations of the inhibition of forward transport by counter-transported ions on the “cis” membrane side for the parallel (A) and serial (B) exchange models. Simulated inhibition of forward transport with ion concentrations as in are given in the top panels, together with data points replotted from . Also scaled to the simulations are data from mentioned in the text. Bottom panels give the simulated inhibition of forward transport by cytoplasmic Na with 140 mM of extracellular Na, an extracellular pH of 8.2, and cytoplasmic pH values of 6.0, 6.8, 7.2, and 7.6. The data points are from experiments described previously for a cytoplasmic pH of 6.8 ().

Simulations of reverse mode activity for the parallel (A) and serial (B) exchange models. Simulated extracellular pH dependence of NHE activity at cytoplasmic Na concentrations of 140 and 10 mM are given in the top panels with data points replotted from and scaled to the simulations. Simulated cytoplasmic Na dependence of reverse NHE activity at extracellular pH values of 6.0 and 8.2 are given in the bottom panels together with data points from for the cytoplasmic Na dependence of reverse NHE1 activity, fitted to a Hill equation with a slope coefficient of 2.

Extracellular Na dependence for native NHE3 in OK cells and native NHE1 in mouse NHE1 wild-type skin fibroblasts. (A) Extracellular Na dependence of native NHE3-induced proton fluxes at intracellular pH (pH_i) 6.0 (left) and 7.2 (right) in OK cells. Note biphasic shape of Na dependence of NHE3 transport at pH_i 6.0 and sigmoidal shape at pH_i 7.2, similar to that observed for NHE1 in CHO cells (see ) and NHE1 wild-type mouse skin fibroblasts (see below). (B) Extracellular Na dependence of native NHE1-induced proton fluxes at intracellular pH (pH_i) 6.0 (left) and 7.2 (right) in wild-type NHE1 mouse skin fibroblasts. Note biphasic shape of Na dependence of NHE1 transport at pH_i 6.0 and sigmoidal shape at pH_i 7.2. Magnitude of Na-induced proton fluxes and extracellular Na dependence are very similar to CHO cells, which express natively NHE1 on the plasma membrane (see ). No proton fluxes were detected in NHE1 knockout mouse fibroblasts or in CHO-derived AP-1 cells, which are devoid of plasmalemmal NHE1 (not depicted).

Extracellular Na and cytoplasmic proton dependence of NHE1 in voltage-clamped CHO cells. (A) Composite data from plotted with standard errors. (B) Average proton fluxes at different Na concentrations plotted in dependence on the cytoplasmic free proton concentration. Lines in the plot represent the best fit of data to a Michaelis-Menton relation. (C) Parameters of the Hill equations fitted to the individual records in . Top plot, Hill coefficients; middle plot, half maximal extracellular Na concentrations; bottom plot, maximal proton fluxes. The open circle gives the Hill coefficient for the maximal fluxes extrapolated in B for the different Na concentration (i.e., the bottom plot in D). (D) Extracellular Na dependence of parameters from the Michaelis-Menton fits given in B. Top plot, half-maximal cytoplasmic proton concentration determined for different extracellular Na concentrations; bottom plot, extrapolated maximal proton fluxes.

Detection of NHE1-induced proton fluxes by oscillating an extracellular pH microelectrode. (A) The cell is held in whole cell configuration with its edge 5 μm from the tip of a pH microelectrode, and it is manually oscillated laterally by 50 μm to detect pH gradients. Both bath and pipette solution can be changed rapidly, the latter by the intra-pipette perfusion technique (see Materials and methods). (B) Example of a recording set for NHE1 transport activity in a single CHO cell using different bath sodium concentrations at fixed extracellular (8.2) and intracellular pH (6.0). Black bars mark time points when the cell was moved away from the pH microelectrode.

Simulations of forward transport by a single NHE1 dimer using the parallel (A) and serial (B) coupling models. The top panels give the simulated extracellular Na dependence of NHE activity at cytoplasmic pH values of 6.0, 6.8, 7.2, and 7.6. Data points are from the insets in . Bottom panels give the simulated cytoplasmic pH dependencies of NHE activity at extracellular Na concentrations of 140, 40, 20, 10, and 5 mM. Data points are replotted from the linear proton concentration plot in and scaled to the simulations. For the parallel model in A, the contributions of Mode 1 and Mode 2 to total proton transport are given.

The extracellular Na dependence of proton flux mediated by NHE1 in 25 CHO cells with a cytoplasmic pH of 6.0 (eight cells), 6.8 (five cells), 7.2 (six cells), and 7.6 (six cells). Data points in each experiment represent the average of 5–20 flux determinations. The lines connect the calculated values from the best fits of data points to Hill equations. The insets illustrate the two strikingly different wave forms obtained at pH 6.0 and 7.2 with the corresponding best fits to Hill equations. At pH 6.0, the extracellular Na dependence often appeared biphasic, showing an apparent high affinity component, such that the overall Hill slope <1. At pH 7.2, the extracellular Na concentration dependence of proton flux showed cooperativity with Hill slopes of ∼2. Composite data shown in and document the significance of these differences.

Summary of apparent discrepancies between this study and previous studies of NHE. (A) Symbols reiterate the cytoplasmic pH dependence obtained with 140 mM of extracellular Na from composite data using different cells (open squares) and from experiments in which the cytoplasmic pH was changed (open circles). Simulated results are given as gray lines for 140 and 2 mM of extracellular Na, whereby the fluxes are scaled and plotted as a percentage of maximum. Dotted lines give the range of “typical” pH–flux relations from the literature. (B) A simple Michaelis-Menton curve is given as pseudo-data points. The line that fits the data points is the sum of two Hill equations with slope coefficients of two, as given in the figure. (C) Data points give the maximal proton fluxes obtained from analysis of the cytoplasmic proton concentration dependence of forward NHE activity in . The curve describing the relation is the sum of two Hill equations with slope coefficients of two, as given in the figure.

Cartoons of two models of NHE1 function as a “coupled dimer” that can carry out 2Na/2H exchange. A “parallel” model is shown in A and a “serial” model is shown in B. The upper cartoons in each panel show the assumed ion translocation reactions, whereby binding of either Na ions or protons enables the same translocation reactions of both ions with the same rates. The lower cartoons show the relevant ion binding schemes, whereby Na ions and protons bind competitively to one or two sites. (A) Parallel model. In this model, it is assumed that monomers carry out Na/H exchange activity independently at low cytoplasmic pH (Mode 1). As cytoplasmic pH rises, proton dissociation from regulatory sites favors the development of an interaction between monomers, such that they become coupled to translocate two ions in a parallel or symport fashion (Mode 2). The ion binding scheme is a simple competition of Na and protons for a single site. (B) Serial model. Each monomer of the dimer can bind two substrates, and ion translocation requires occupation of both sites. Each monomer can translocate ions and allows them to dissociate on the opposite membrane side, but the rates of translocation (Kxw and Kwx) are higher when binding sites of both monomers are open to one membrane side (E1 and E2), Thus, transport reactions become coupled in a serial fashion: Translocation of two ions in one direction promotes the translocation of two ions in the opposite direction. The ion binding scheme for each monomer assumes that two protons or Na ions can bind in random order and that all configurations with two ions bound can undergo conformational changes that allow ion dissociation on the opposite membrane side.