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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

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Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti

Ashok K. Pullikuth^1,*,, Karlygash Aimanova^1,*, Wanyoike Kang'ethe², Heather R. Sanders^1,3, and Sarjeet S. Gill^1,2

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

View larger version (162K): [in a new window]   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.

View larger version (62K): [in a new window]   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).

View larger version (67K): [in a new window]   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).

View larger version (86K): [in a new window]   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).

View larger version (26K): [in a new window]   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.

View larger version (29K): [in a new window]   Fig. 6. Functional characterization of AeNHE3 in a NHE-deficient cell line. (A) Untransfected PS120 cells fail to recover intracellular pH following an acid load, but maintain a near neutral pH in culture medium (DMEM). Cells were challenged with a H+ load (+) or left unchallenged (-) and subsequently changed to buffer (135 mmol l-1 NaCl, HBS, pH 7.4) or culture medium (DMEM) and assayed by ratiometric fluorimetry. (B) Polyclonal cells from two separate transfection experiments (NHE3a and NHE3b) expressing full-length AeNHE3 were subjected to acid load and recovery of intracellular pH was monitored by BCECF fluorescence. (C) A stable clone (clone A2) expressing AeNHE3 was subjected to acid load and changed to buffers containing the indicated concentrations of ions to monitor change in intracellular pH (pHi) with (+) or without (-) inhibitors of Na+/K+-ATPase (1 mmol l-1 ouabain) and Na+/K+/Cl- cotransporter (100 µmol l-1 bumetanide). Experiments with another stable clone (clone B3) produced similar results. (D) The cytoplasmic carboxy terminal of AeNHE3 is not required for its function. A stable clone (NHE C-clone A8) that expresses AeNHE C (lacking carboxy 448 amino acids) was assayed for pHi recovery after an acid load in the presence or absence of inhibitors as stated in C. Experiments with another stable line (NHE C-clone B7) produced similar results. The buffers also contained 5 mmol l-1 glucose, 2 mmol l-1 CaCl2 and 1 mmol l-1 MgCl2. Values are means ± standard error (N=4-8).

View larger version (17K): [in a new window]   Fig. 7. Amiloride-insensitive 22Na+-uptake in AeNHE3-expressing fibroblast cell line. Full-length (NHE3FL) or carboxy-terminal truncated (NHE3C) 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.).