Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger.

Abstract

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H + exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H + and amiloride appear to compete with Na + for binding to this site. The apparent affinity for extracellular Na § (Na~) and amiloride was independent of intracellular H + (Hi+), Nai +, or an outwardly directed H + gradient. The interaction of H + with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of ~2. The apparent affinity for Hi+ was independent of Na~ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H § gradient across the membrane. Nai+/Ho + exchange (reverse mode) is an electroneutral process with a Na+/H + stoichiometry of 1. The dependence of Na~+/H 2 exchange on Na~ + was sigmoidal, with a Hill coefficient of 2.16. Nai + competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai + decreased with the increase in the outwardly directed H § gradient. High H 2 inhibited exchange activity in the reverse mode. We conclude that intracellular Na § and H § can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na + and H + are determined by the direction and magnitude of the H § gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H § exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.

Sodium‐hydrogen exchange in guinea‐pig ventricular muscle during exposure to hyperosmolar solutions.

“…The high a'a might have inhibited Na+-H+ exchange-mediated pHi recovery because Na+ competes with H+ at the intracellular transport site of the exchanger (Grinstein et al 1984;Green, Yamaguchi, Kleeman & Muallem, 1988b). In addition, a high a',a should enhance cellular acid generation and thus cause a decrease in the apparent rate of Na+-H+ exchange-mediated pH1 recovery (Kaila & Vaughan-Jones, 1987).…”

Extracellular H+ inactivation of Na(+)‐H+ exchange in the sheep cardiac Purkinje fibre.

“…Jean et al (1985) propose that, in brain synaptosomes, the transmembrane H+ ratio (i.e. pH0-pHj) is the important determinant of acid extrusion and Green et al (1988) extend this proposal by suggesting that, in osteoblasts, the exchanger can extrude acid only if the H+ gradient is outwardly directed. Both of the above proposals do not apply to Na+-H+ exchange in the cardiac Purkinje fibre and given the general similarity of the exchanger in the three preparations, it seems likely that the proposals do not apply in the other cell types either.…”

“…This indicates that inhibition is proportional to [H+]no where n, on average, equals 1-2, suggesting that one (but possibly > 1) H+ ion binds to an external site in order to promote inhibition. Further (Aronson, Suhm & Nee, 1983), lymphocytes (Grinstein, Cohen & Rothstein, 1984) and cultured osteoblasts (Green, Yamaguchi, Kleeman & Muallem, 1988).…”

Steady-state Function of the Ubiquitous Mammalian Na/H Exchanger (NHE1) in Relation to Dimer Coupling Models with 2Na/2H Stoichiometry

“…As pointed out in the Introduction, a simple transport function of mammalian NHEs ( Kinsella and Aronson, 1982 ;Jean et al, 1985 ;Schmalzing et al, 1986 ;Semplicini et al, 1989 ;Doktor et al, 1991 ;Levine et al, 1993 ;Demaurex et al, 1995 ;Wu and Vaughan-Jones, 1997 ;Slepkov et al, 2007 ) was challenged by multiple previous studies that showed cooperative activating effects of Na ( Green et al, 1988 ;Otsu et al, 1989Otsu et al, , 1993. On the basis of the present work and the cited articles, it is established that the Na dependence of exchange activity can be cooperative from both membrane sides.…”

“…First, attempts to defi ne pre -steady-state kinetics of NHE3 at low temperatures revealed a highly cooperative activation by Na that was suggested to refl ect dimer coupling ( Otsu et al, 1989( Otsu et al, , 1993. Second, activation of reverse Na/H exchange by cytoplasmic Na is cooperative with a Hill coeffi cient close to 2 ( Green et al, 1988 ). Third, analysis of NHE1 mutants with altered regulatory function suggests that the ion dependencies of transport per se are more steep Cartoons of two models of NHE1 function as a " coupled dimer " that can carry out 2Na/2H exchange.…”