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 Na-22(+) efflux from Na-22(+)-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 Na-22(+) 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 (pH(i)) dependence of Na-22(+) efflux roughly exhibited a bell-shaped profile; mild acidification from pH(i) 7.5 to 7 dramatically accelerated Na-22(+) efflux, whereas acidification from pH(i) 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive Na-22(+) efflux. Cell ATP depletion and mutation of NHE1 at Arg(440) (R440D) caused a large acidic shift of the pH(i) profile for Na-22(+) efflux, whereas mutation at Gly(455) (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.