Responsiveness of mutants of the nhe-1 isoform of the na+/h+
antiport to osmotic stress.
Bianchini, Laurence, Andras Kapus, Gergely Lukacs, Sanjeev Wasan,
Shigeo Wakabayashi, Jacques Pouyssegur, Frank H. Yu, John Orlowski,
and Sergio Grinstein.
Division of Cell Biology, Hospital for Sick Children, 555
University Ave.
APStracts 2:0228C, 1995.
Hypertonic activation of NHE-1, the ubiquitous Na+/H+ exchanger, plays
a central role in cell volume regulation, yet little is known about
the underlying mechanism. We probed the osmotic responsiveness of
full-length and truncated constructs of NHE-1 transfected into cells
lacking endogenous antiport activity. The hypertonic stimulation of
NHE-1 was preserved following heterologous transfection of the full
-length NHE-1 or of constructs truncated at positions 698 or 703. In
contrast, mutants truncated at position 635 (_635) failed to respond
to osmotic challenge. _635 transfectants behaved as if constitutively
activated, having a permanently elevated cytosolic pH (pHi) under
isotonic, unstimulated conditions. The _635 mutant displayed H+
binding with high affinity and low cooperativity. Constructs _582 or
_566 had a reduced H+ sensitivity and were therefore inactive at
resting pHi. Such cells were unresponsive to osmotic stress near
physiological pHi, but could be activated by shrinking following 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 likely
controlled by different regions of the molecule. The data indicate
that volume or osmolarity-sensitive site(s) exist between the N
-terminus and residue 566. This putative volume-sensitive site is
therefore different from the site(s) postulated to mediate the
stimulatory effects of calcium and growth factors.
Received 6 January 1995; accepted in final form 12 April 1995.
APS Manuscript Number C12-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 July 1995.