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Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry.

Fuster D, Moe OW, Hilgemann DW - J. Gen. Physiol. (2008)

Bottom Line: The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models.For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na.We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Department of Internal Medicine, University of Texas-Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1(-/-) skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K(1/2)) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K(1/2) for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

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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.
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fig2: 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.

Mentions: The following solutions were used in Figs. 2–6: bath solution: 0–140 NaCl, 2 CaCl2, 1 MgCl2, 0.1 Tris, pH 8.2; pipette solution: 60 KOH, 30 l-aspartate acid, 10 KCl, 1 EGTA, 0.5 MgCl2, 10 MgATP, and 50 Mes, pH 6.0, 50 Pipes, pH 6.8, 50 Mops, pH 7.2, or 50 HEPES, pH 7.6 (7.6).


Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry.

Fuster D, Moe OW, Hilgemann DW - J. Gen. Physiol. (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2553392&req=5

fig2: 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.
Mentions: The following solutions were used in Figs. 2–6: bath solution: 0–140 NaCl, 2 CaCl2, 1 MgCl2, 0.1 Tris, pH 8.2; pipette solution: 60 KOH, 30 l-aspartate acid, 10 KCl, 1 EGTA, 0.5 MgCl2, 10 MgATP, and 50 Mes, pH 6.0, 50 Pipes, pH 6.8, 50 Mops, pH 7.2, or 50 HEPES, pH 7.6 (7.6).

Bottom Line: The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models.For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na.We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Department of Internal Medicine, University of Texas-Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1(-/-) skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K(1/2)) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K(1/2) for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

Show MeSH
Related in: MedlinePlus