Function and regulation of rat sodium/hydrogen exchanger isoforms

Recent molecular cloning studies have identified multiple isoforms of the Na;1. Functional characterization of the rat NHE2 isoform revealed differences in its pharmacological and kinetic properties compared to two other isoforms, NHE1 and NHE3. Half-maximal inhibition of NHE2 activity by amiloride and some of its analogues occurred at intermediate concentrations compared to NHE1 and NHE3, although the rank order of potency of the compounds was similar. Analyses of its kinetic behaviour also showed that while the transport dynamics of NHE2 were similar to those of the other isoforms, it had quite different affinities for its cation substrates. Therefore, these differences provide a biochemical and a pharmacological basis for discriminating NHE2 from NHE1 and NHE3.;2. Structural analysis of the NHE3 isoform was conducted in two studies by generating deletion and chimeric mutants. The first study examined the structural domains involved in its regulation by cAMP and ATP. Chimeras of NHE1 and NHE3 that were constructed by the homologous exchange of their C-terminal cytosolic tail regions demonstrated that the cytosolic tail of NHE3 is sufficient to confer sensitivity to inhibition by cAMP. Additional analysis of NHE3 deletion mutants revealed a region between amino acids 579 and 684 that is essential for this inhibitory effect. In contrast, comparable ATP dependence was observed in wild type and all mutant exchangers examined suggesting that a region of the molecule in or adjacent to the transmembrane domain is involved in this response. It is concluded that different sites, suggesting of different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.;3. In the final study, NHE3 inhibition by hyperosmolarity was examined using the same mutational approach. The cytoplasmic tail of NHE3 was insufficient to confer the hyperosmotic inhibitory effect to a chimeric exchanger that contained the N-terminal transmembrane region of NHE1, suggesting that an interaction between the N and C termini of NHE3 may be required for this inhibition. Deletion analysis revealed two hyperosmolarity sensitive domains in the NHE3 cytosolic tail region: a hyperosmotically activatable region between amino acids 579 and 638 and an inhibitory region between amino acids 649 and 684, the latter acting in a dominant manner in the presence of the former domain. These data are consistent with the conclusion that hyperosmotic inhibition of NHE3 is mediated by mechanisms that appear to be distinct from those that underlie cAMP-sensitivity and ATP dependence.