First published online August 30, 2006
Journal of Experimental Biology 209, 3529-3544 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02419
Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti
Ashok K. Pullikuth1,*,
,
Karlygash Aimanova1,*,
Wanyoike Kang'ethe2,
Heather R. Sanders1,3,
and
Sarjeet S. Gill1,2
1 Department of Cell Biology and Neuroscience, University of California,
Riverside, CA 92521-0146, USA
2 Graduate Program in Environmental Toxicology, University of California,
Riverside, CA 92521-0146, USA
3 Graduate Program in Microbiology, University of California, Riverside, CA
92521-0146, USA
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Fig. 1. Sequence and topology of the sodium/proton exchanger, NHE3 from Aedes
aegypti. (A) Canonical translation of Aedes NHE3 (AF187723)
aligned with Anopheles NHE3 (AY170874) and their close relatives in
Drosophila, crab and vertebrates. DmNHE3a (AAF60313) refers to the
full-length Drosophila NHE3 homolog, whereas DmNHE3b (AY048581) and
DmNHE3c (AY128467) refer to possible splice variants identified by
high-throughput cDNA sequencing and by RT-PCR analysis, respectively.
Identical residues are shaded in red and conserved residues are boxed. DmNHE2
from the original annotation (Giannakou
and Dow, 2001 ) is here assigned to the NHE3 family.
AeNHE3 ORF is encoded by 21 exons; asterisks indicate the position of
introns. (B) Predicted transmembrane topology and potential phosphorylation
sites in AeNHE3. The positions of residues at the beginning and end
of predicted transmembrane helices are numbered. Putative phosphorylation
sites and ERK-D domains are given in green and blue, respectively. (C)
Comparison of amiloride binding sites in NHEs. A critical leucine (arrow)
within the pocket renders amiloride sensitivity to vertebrate NHE1, 2 and 4.
The corresponding residue in AeNHE3 is phenylalanine (F313).
Substitution of leucine to phenylalanine in vertebrate NHE1 and 2 removes the
amiloride sensitivity in these NHEs (see Discussion). Conserved residues are
shown in red whereas semi-conservative substitutions are back-shadowed in
blue. (D) Calcineurin B homologous protein (CHP) binding site is conserved in
insect NHE3. The conserved hydrophobic residues (green) are required for
efficient transport function of plasma membrane NHEs. Note that the shorter
AeNHE3 (AeNHE3-2.8kb) contains only half-site for CHP binding. Ae, Ag
and Hs refer to Aedes aegypti, Anopheles gambiae and human,
respectively. Accession numbers (GenBank; right) and amino acid positions
(left) are indicated.
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Fig. 2. Expression and localization of AeNHE3 in osmoregulatory organs in
larva and adult Aedes aegypti. AeNHE3 is highly expressed in the
posterior midgut (PMG) of larval (A) and adult (C) Aedes aegypti.
Malpighian tubules (MT) and hindgut (HG) also show high AeNHE3
immunoreactivity (red). In larva, anterior midgut (AMG) expresses lower levels
of NHE3 whereas adult AMG expresses levels comparable to PMG. Interestingly,
the distal part of adult PMG shows reduced expression of NHE3. In both
anterior (D) and posterior (E) midgut, NHE3 expression (red) is restricted to
the basolateral membrane. Actin in D and E is stained green. (B) The
background fluorescence with preimmune serum used as primary antibody. Immune
antibody preabsorbed with the peptide used for immunization gave similar
results as in B. Scale bars, 100 µm (B); 50 µm (D,E).
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Fig. 3. Expression of NHE3, Na+/K+-ATPase and V-ATPase in the
gastric caeca of larval Aedes aegypti. AeNHE3 is highly expressed in
the distal segment of gastric caeca (A) predominantly in the basolateral
membrane (orange in B) of distal segment. Only faint labeling of NHE3 was
found in the proximal segment of caeca (C, arrow).
Na+/K+-ATPase is excluded from regions of higher NHE3
expression but is enriched in proximal segment (yellow in E) and is localized
to the basolateral membrane (yellow in F). V-ATPase localizes to the apical
plasma membrane of both proximal and distal parts of the gastric caeca (H,I).
Weaker V-ATPase labeling was found in the distal basal membrane (arrow in H).
Gastric caeca was labeled with anti-NHE3 antibody alone (A-C) (red in A,
orange in B,C) or together (red in E-G) with
anti-Na+/K+-ATPase monoclonal antibody (yellow in E-G).
To visualize structures, actin was labeled with Alexa Fluor 488-phalloidin in
B-D (green). Expression of the V-ATPase was detected with polyclonal
antibodies to the B-subunit (H,I). Preimmune serum was used as negative
control (D) with phalloidin (green). Immunohistochemistry was performed on
whole mounts (A,E) or tissue sections as described in Materials and methods.
E1 (red for NHE3) and E2 (yellow for Na+/K+-ATPase) show
the separate channels that are merged in E. F and G are similarly merged
images of dual labeling with NHE 3 (red) and
Na+/K+-ATPase (yellow) antibodies. Scale bars, 100 µm
(E,E1,E2); 50 µm (A-D,F-I).
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Fig. 4. Expression of AeNHE3, Na+/K+-ATPase and
V-ATPase in the Malpighian tubules and hindgut of Aedes aegypti.
Whole-mount (A,G) or paraffin sections (B-F) were analyzed by labeling with
V-ATPase B-subunit (A,B), anti-NHE3 antibody (D-G) and
anti-Na+/K+-ATPase  1 subunit monoclonal antibody
(E-G). Preimmune serum was used as negative control (C) with phalloidin
(green), and similar results were obtained with peptide preabsorbed anti-NHE3
antibody. V-ATPase is primarily localized to the apical membrane of principal
cells (PC in A,B) whereas both NHE3 (D,F) and
Na+/K+-ATPase (F) localize to basal aspect of the plasma
membrane of principal cells. In median Malpighian tubules NHE3 expression was
also detected in the apical membrane (E, arrow) and in the basal membrane
colocalizing with Na+/K+-ATPase (E, arrowhead). Weaker
expression of NHE3 was also detected as punctate structures in the cytoplasm
of Malpighian tubules (F,F1). Anterior hindgut and Malpighian tubules exhibit
similar expression levels of NHE3, whereas in the rectum both NHE3 and
Na+/K+-ATPase are remarkably enhanced (G,G1,G2).
E1,F1,G1 show NHE3 expression (red), while E2,F2,G2 depict
Na+/K+-ATPase expression (yellow), and merged channels
depicting co-localization of NHE3 and Na+/K+-ATPase are
shown in E-G. Scale bars, 50 µm (A-F); 100 µm (G).
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Fig. 5. AeNHE3 rescues S. cerevisiae NHA1 function when
heterologously expressed in mutant cells lacking endogenous Na+
efflux and Na+/H+ exchanger proteins. Yeast cells were
grown in alkali cation free-AP medium overnight, and cell density adjusted to
an OD600=1. Tenfold serial decimal dilutions were spotted on: (A)
AP plates containing 50 mmol l-1 NaCl, and (B) minimal media plates
containing 100 µg ml-1 Hygromycin B. Growth was recorded after 4
days in (A), and 3 days in (B). Strain G19 was used as the NHA1, NHX1
wild-type control; nha1, nhx1 is the AXT3 strain. AeNHE3
expression was under galactose induction; all controls were transformed with
an empty vector.
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Fig. 7. Amiloride-insensitive 22Na+-uptake in
AeNHE3-expressing fibroblast cell line. Full-length (NHE3FL) or
carboxy-terminal truncated (NHE3 C) AeNHE3 were transiently
transfected into PS120 cells. 3-4 days after transfection, cells were assayed
for 22Na+ uptake in the presence of inhibitors (1 mmol
l-1 amiloride or 100 µmol l-1 EIPA). Values are
d.p.m. mg-1 protein min-1 (means ± s.e.m.).
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© The Company of Biologists Ltd 2006
