RESPONSIVENESS OF MUTANTS OF NHE1 ISOFORM OF NA+ H+ ANTIPORT TO OSMOTIC-STRESS/

Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about th e underlying mechanism. We probed the osmotic responsiveness of fullle ngth and truncated constructs of NHE1 transfected into cells lacking e ndogenous antiport activity. The hg pertonic stimulation of NHE1 was p reserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants tr uncated at position 635 (Delta 635) failed to respond to osmotic chall enge. Transfectants (Delta 635) behaved as if constitutively activated , having a permanently elevated cytosolic pH (pH(i)) under isotonic, u nstimulated conditions. The Delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs Delta 582 or Delta 56 6 had a reduced H+ sensitivity and were therefore inactive at resting pH(i). Such cells were unresponsive to osmotic stress near physiologic al pHi but could be activated by shrinking after an acid load. Jointly , these results suggest that the H+ affinity and high cooperativity of the antiporter, earlier attributed to a single ''modifier site,'' can be varied independently and are probably controlled by different regi ons of the molecule. The data indicate that volume or osmolarity-sensi tive site(s) erdst between the NH2-terminus and residue 566. This puta tive volume-sensitive site is therefore different from the site(s) pos tulated to mediate the stimulatory effects of calcium and growth facto rs.