Kinetic Dissection of Two Distinct Proton Binding Sites in Na+/H+ Exchangers by Measurement of Reverse Mode Reaction

Shigeo Wakabayashi,Takashi Hisamitsu,Tianxiang Pang,Munekazu Shigekawa

doi:10.1074/jbc.m306690200

Shigeo Wakabayashi, Takashi Hisamitsu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m306690200

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Abstract

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.

Highlights

The Naϩ/Hϩ exchangers (NHEs)1 belong to one of the secondary active transporter families that catalyze the transport of ions or solutes using a driving force generated by active ion pumps
We examined the effect of intracellular acidification on the reverse mode of Na؉/H؉ exchange by measuring 22Na؉ efflux from 22Na؉-loaded PS120 cells expressing the Na؉/H؉ exchanger (NHE) isoforms NHE1, NHE2, and NHE3
We found that 22Naϩ efflux is dramatically stimulated by intracellular acidification but almost completely inhibited by modest alkalinization, which provides a strong piece of evidence for the existence of an intracellular Hϩ modifier site in these NHE isoforms

Summary

EXPERIMENTAL PROCEDURES

Materials—The amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was a gift from New Drug Research Laboratories of Kanebo, Ltd. (Osaka, Japan). 22NaCl was purchased from PerkinElmer Life Sciences. Measurement of 22Naϩ Efflux—Serum-depleted cells in 24-well dishes were loaded with 22Naϩ by preincubating them for 30 min at 37 °C in chloride/KCl medium comprising 20 mM Hepes/Tris (pH 7.4), 0.2–1.2 mM 22NaCl (37 kBq/ml), 1.9 –140 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 5 ␮M nigericin. After removal of the radioactive preincubation solution, 22Naϩ efflux was started by adding the same choline chloride/ KCl medium except that it contained 2 mM ouabain and 100 ␮M bumetanide but not 22Naϩ. For the data at zero time, this efflux solution was not added. Measurement of 22Naϩ Uptake—22Naϩ uptake activity was measured using serum-depleted cells grown in 24-well dishes in the presence or absence of EIPA as described previously [20]

RESULTS

We examined the effect of cell ATP depletion on the pHi

DISCUSSION