Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction.
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.
Wakabayashi
Shigeo
S
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565 Japan. wak@ri.ncvc.go.jp
Hisamitsu
Takashi
T
Pang
Tianxiang
T
Shigekawa
Munekazu
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D011522:Protons; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D020033:Protein Isoforms; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D012964:Sodium