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 i and H l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na+ EXC was measured as Na o -stimulated Na+ efflux and Na+/H+ EXC as Na o -stimulated H+ efflux and ΔpH o -stimulated Na+ influx into acid-loaded cells.
The activation of Na+/Na+ EXC by Na o 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 (K m 2.2 mM) and low affinity (K m 108 mM) sites for a single- or two-carrier system. The activation of Na+/H+ EXC by Na o (pH i 6.6, Na i <1 mM) also showed high (K m 11 mM) and low (K m 248 mM) affinity sites. External H+ competitively inhibited Na+/Na+ EXC at the low affinity Na o site (K H 52 nM) while internally H+ were competitive inhibitors (pK 6.7) at low Na i and allosteric activators (pK 7.0) at high Na i .
Na+/H+ EXC was also inhibited by acid pH o and allosterically activated by H i (pK 6.4). We also established the presence of a Na i regulatory site which activates Na+/H+ and Na+/Na+ EXC modifying the affinity for Na o of both pathways. At low Na i , Na+/Na+ EXC was inhibited by acid pH i and Na+/H+ stimulated but at high Na i , 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 Na o sites,cis-inhibited by external H o , allosterically modified by the binding of H+ to a H i 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+/H+ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH i -stimulated 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 explained by a model of Na+/H+ EXC with several conformational states, H i and Na i 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 Na i 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+.
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Morgan, K., Canessa, M. Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway. J. Membrain Biol. 118, 193–214 (1990). https://doi.org/10.1007/BF01868604
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DOI: https://doi.org/10.1007/BF01868604