DISTINCT STRUCTURAL DOMAINS CONFER CAMP SENSITIVITY AND ATP DEPENDENCE TO THE NA+ H+ EXCHANGER NHE3 ISOFORM/

Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/ H+ exchanger ex pressed at high levels in apical membranes of these ce lls. In contrast, the ubiquitous, housekeeping isoform of the exchange r (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP, To gain insight in to the molecular mechanisms of ATP dependence and cAMP regulation of N HE3, a series of mutations were constructed by progressively truncatin g segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3 Delta 684, NHE3 Del ta 638, and NHE3 Delta 579). In addition, chimeric antiporters were co nstructed with the N-terminal transmembrane domain of NHE3 linked to t he entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (c himera NHE1/3). These constructs were heterologously expressed in anti port-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radio isotope determinations of Na+ influx. Forskolin, which directly stimul ates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3 /1, suggesting that the cytoplasmic domain of NHE3 was sufficient to c onfer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3 Delta 684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3 Delta 638 and more profound truncations (NHE3 Delta 579) obliterated t he effect of forskolin. These findings suggest that a region found bet ween amino acids 579 and 684 is essential for the cAMP response of NHE 3. In contrast, comparable ATP dependence was observed in all exchange r constructs examined, These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transm embrane domain, which is most conserved between isoforms. It is conclu ded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.