J. Membrane Biol. 118, 193-214 (1990) The dournal of Membrane Biolocjy 9 Springer-Verlag New York Inc. 1990 Interactions of External and Internal H + and Na + with Na+/Na + and Na+/H + Exchange of Rabbit Red Cells: Evidence for a Common Pathway Kevin Morgan and Mitzy Canessa Endocrine-Hypertension Division, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 Summary. We have studied the kinetic properties of rabbit red cell (RRBC) Na*/Na + and Na+/H + exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na + and H + at the internal (i) and external (o) sites for both exchanges modes, RRBC containing varying Na~ and Hi were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na + EXC was measured as Nao-stimulated Na + efflux and Na+/H + EXC as Nao-stimulated H + efflux and ApHo-stimu- lated Na + influx into acid-loaded cells. The activation of Na+/Na + EXC by Nao at pH i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (Km 2.2 mM) and low affinity (Kin 108 raM) sites for a single- or two-carrier system. The activation of Na+/ H + EXC by Na o (pHi 6.6, Nai < 1 mM) also showed high (Kin 11 mM) and low (Km 248 raM) affinity sites. External H + competi- tively inhibited Na+/Na + EXC at the low affinity Nao site (KH 52 riM) while internally H + were competitive inhibitors (pK 6.7) at low Nai and allosteric activators (pK 7.0) at high Nai. Na+/H + EXC was also inhibited by acid pHo and allosteri- cally activated by Hi (pK 6.4). We also established the presence of a Nai regulatory site which activates Na+/H + and Na§ + EXC modifying the affinity for Na o of both pathways. At low Nai, Na+/Na + EXC was inhibited by acid pHi and Na+/H * stimulated but at high Nai, Na+/Na + EXC was stimulated and Na+/H + inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Nao sites, cis- inhibited by external Ho, allosterically modified by the binding of H + to a Hi regulatory site and regulated by Na i. These findings are consistent with Na+/Na + EXC being a mode of operation of the Na+/H+exchanger. Na § + EXC was partially inhibited (80-100%) by dimeth- yl-amiloride (DMA) but basal or pHi-stimalated Na+/Na + EXC (pH i 6.5, Na i 80 mM) was completely insensitive indicating that Na-/Na + EXC is an amiloride-insensitive component of Na+/ H + EXC. However, Na* and H + efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA; this also indicates that the Na+/H + EXC might operate in reverse or uncoupled modes in the absence of Na+/Na + EXC. In summary, the observed kinetic properties can be ex- plained by a model of Na +/H § EXC with several conformational states, Hi and Nai regulatory sites and loaded/unloaded internal and external transport sites at which Na § and H + can compete. The occupancy of the H + regulatory site induces a conformational change and the occupancy of the Nai regulatory site modulates the flow through both pathways so that it will conduct Na+/ H + and/or Na+/Na + EXC depending on the ratio of internal Na* : H + . Key Words rabbit red cells 9 Na/Na exchange 9 Na/H ex- change 9 H + and Na + interactions I. Introduction Rabbit red blood cells are similar to human erythro- cytes with respect to the mechanisms present within their cell membranes for the translocation of Na +. Both species exhibit a ouabain-sensitive Na + pump (Villamil & Kleeman, 1959; Glynn & Karlish, 1975) together with ouabain-insensitive Na+/Na § EXC which is also capable of transporting Li + (Haas, Schooler & Tosteson, 1975; Duhm & Becker, 1979) and can be inhibited by phloretin (Pandey et al., 1978). The maximal velocity (Vm~x) of Na+/Li § countertransport in human red cells is of low magni- tude (Haas et al., 1975; Canessa et al., 1980) and it has been assumed that in-vivo it operates an obliga- tory one-for-one Na +/Na + EXC. In rabbit red cells, the Vma• of this system is at least 10-fold higher than in human erythrocytes but only partially blocked by phloretin (Duhm & Becker, 1979). Several studies have confirmed that the activity of the Li+/Na + EXC is elevated in red cells of pa- tients with essential hypertension and it is geneti- cally determined (Canessa et al., 1980; for reviews, Canessa, 1984; Canessa, Brugnara & Escobales, 1987; Canessa, Morgan & Semplicini, 1988). Aron- son (1982), Sachs, Faler and Rabson (1982) and Funder et al. (1984) have suggested that Na+/Na +, Na+/Li § EXC might operate as a Na§ § EXC, a transport system involved in proximal tubular reab- sorption (Murer, Hopfer & Kinne, 1976; Aronson &