Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines
Josette Noel
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Danile Roux
0
Jacques Pouyssgur
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0
Centre de Biochimie-CNRS, Universite de Nice
,
Parc Valrose, 06108, Nice
,
France
*These authors have contributed equally to this work Author for correspondence (e-mail: )
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Na+/H+ exchangers (NHEs) are transporters that exchange
sodium and proton ions across the plasma membrane at the
expense of their chemical gradient. In higher eukaryotes
these transporters exist as multiple specialized isoforms.
For example, NHE1, the ubiquitously expressed form is a
major pH-regulating system whereas the epithelial NHE3
isoform is specialized in transepithelial Na+ transport.
NHE1 and NHE3 can be very well distinguished
pharmacologically with the HOE694 specific inhibitor and
immunologically with specific polyclonal and monoclonal
antibodies. With these molecular tools we investigated the
specific steady state expression of the two NHE isoforms in
polarized epithelial cells in culture.
Endogenous NHE3 in OK cells or NHE3-VSVG
transfected in either OK or MDCK cells showed an exclusive
expression of the transporter at the apical membrane.
Overexpression of NHE3 did not result in any spill over on
the basal lateral side. These results obtained by functional
measurement of NHE3 activity were fully consistent with
its detection only at the apical side by immunofluorescence
The Na+/H+ exchanger (NHE) is a plasma membrane protein that
functions in higher eukaryotic cells primarily for the maintenance
of intracellular pH but also contributes to the transepithelial
transport of Na+ (for reviews see Grinstein et al., 1989; Tse et al.,
1993a; Nol and Pouyssgur, 1995). H+ extrusion is ensured by
the extreme sensitivity of NHE to intracellular H+ and by the
inwarded-directed Na+ gradient established by the Na+, K+
ATPase. NHE is therefore powerful for restoring intracellular pH
towards neutrality in response to acid-load insults (Pouyssgur et
al., 1984). In intestine and renal epithelial cells, NHE, expressed
at the luminal side contributes primarily in transepithelial
transport of Na+, the plurality of functions being dictated by the
location and the kinetic parameters of the NHE molecules.
The first NHE to be cloned and referred to as NHE1 is a
glycoprotein of 110 kDa (Sardet et al., 1989, 1990) that exists in
the membrane as a homodimer (Fafournoux et al., 1994). The
N-terminal half of the molecule possesses multiple
transmembrane segments (10 to 12) and constitutes the transporter unit.
and confocal microscopy. By contrast, using the same cells,
the same culture conditions and the same detection
methods, we clearly detected NHE1 at both specialized
membranes of four different polarized epithelial cell lines.
Furthermore, biotinylation of cell surface proteins of
MDCK, OK and HT-29 cells followed by
immunoprecipitation of NHE1 revealed expression of the transporter at
both sides of the polarized epithelial cells. Interestingly, the
cell surface expression correlated well with the
corresponding NHE1 activities. In addition, immunodetection by
fluorescence microscopy was found to be qualitatively
consistent with the above-reported results.
We therefore conclude that the epithelial and more
specialized NHE3 isoform is exclusively restricted to the apical
side of epithelial cells. In marked contrast, both
endogenous or ectopically expressed NHE1 isoform, have the
capacity to be expressed in both the apical and basal lateral
membranes of polarized cells in cultures.
The C-terminal cytoplasmic domain of about 300 amino acids,
although dispensable for Na+/H+ exchange activity, controls the
affinity for intracellular H+ and represents a regulatory domain
referred to as the transducer unit. Indeed, partial or complete
removal of the cytoplasmic domain reduces or abolishes NHE
activation in response to various extracellular stimuli that
include growth factors, hormones, fibronectin, hyperosmotic
shock (Wakabayashi et al., 1992, 1994; Bianchini et al., 1995;
C. Lechene and J. Pouyssgur, unpublished results). NHE, like
essential proteins, and as previously predicted (Clark and
Limbird, 1991), exists as a multigene family. Five isoforms
encoded by distinct genes have so far been identified (Sardet et
al., 1989; Orlowski et al., 1992; Tse et al., 1992, 1993b; Klanke
et al., 1995). NHE1-4 possess the generic NHE1 hydropathy
profile, and share between them about 40% identity at the amino
acid level, with a stronger conservation in the transporter unit
(Tse et al., 1993a; Nol and Pouyssgur, 1995). Whereas NHE1
is virtually expressed in all tissues, NHE 2, 3 and 4 have
restricted expression to epithelial tissues and restricted
expression within polarized cells. For instance, the location of
NHE2 and NHE3 at the apical surface of epithelial cells confers
a specialized function to these isoforms, pointing out the
importance of the mechanisms involved in membrane protein sorting.
A large effort has been devoted to this question over the last
decade and although considerable progress and unders (...truncated)