Am J Physiol Renal Physiol 296: F730–F750, 2009.
First published February 4, 2009; doi:10.1152/ajprenal.90564.2008.
NHE8 is an intracellular cation/H⫹ exchanger in renal tubules
of the yellow fever mosquito Aedes aegypti
Peter M. Piermarini,1 Dirk Weihrauch,2 Heiko Meyer,2 Markus Huss,2 and Klaus W. Beyenbach1
1
Department of Biomedical Sciences, Cornell University, Ithaca, New York; and 2Department of Biology, University
of Osnabrück, Osnabrück, Germany
Submitted 19 September 2008; accepted in final form 27 January 2009
Na/H exchange; Malpighian tubules; Xenopus laevis oocytes; electrophysiology
of insects actively secrete fluid
to form urine. In the yellow fever mosquito Aedes aegypti
(referred to as Aedes hereafter), the blind-ended (distal) segments of the Malpighian tubules transport both K⫹ and Na⫹
actively from the extracellular fluid (hemolymph) to the tubule
lumen, with Cl⫺ and water following their electrochemical and
osmotic gradients, respectively (6, 8, 18). The primary urine
then flows through the proximal segments of the tubule and
into the distal digestive tract (hindgut and rectum), where the
urine may be modified before its excretion.
Urine formation in distal segments of Aedes Malpighian
tubules is primarily mediated by principal cells, which are the
majority cell type of the tubule in terms of both abundance and
mass (6, 15, 65). In Malpighian tubules of dipteran insects,
THE RENAL (MALPIGHIAN) TUBULES
Present address of D. Weihrauch: Dept. of Biological Sciences, Univ. of
Manitoba, Winnipeg R3T 2N2, Canada.
Address for reprint requests and other correspondence: P. M. Piermarini,
Cornell Univ., College of Veterinary Medicine, Dept. of Biomedical Sciences,
Ithaca, NY 14853 (e-mail: [email protected]).
F730
such as Aedes and Drosophila melanogaster (hereafter referred
to as Drosophila), the apical membrane of principal cells forms
an elaborate, luminal brush border, with each microvillus
housing a mitochondrion (5, 6, 8). The brush border is enriched
with the V-type H⫹-ATPase (24, 55, 59, 81), which is thought
to establish all of the electrochemical potentials that drive
transcellular and paracellular electrolyte transport (5, 7).
The mechanisms that mediate the extrusion of K⫹ and Na⫹
across the apical membrane of principal cells have not yet been
elucidated. Current models propose that a monovalent cation/H⫹ exchanger recycles luminal protons, pumped by the
V-type H⫹-ATPase, in exchange for the extrusion of intracellular K⫹ and/or Na⫹ (6, 8, 18, 47, 60), analogous to the model
proposed for the secretion of K⫹ (i.e., alkalinization) across the
midgut of the tobacco hornworm Manduca sexta (31). The
molecular identification of this exchanger has eluded many
investigators. Present hypotheses consider the exchanger to be
related to the cation proton antiporter (CPA) superfamily,
which includes the CPA1 or solute carrier 9 (SLC9) family of
Na/H exchangers (NHEs) and the CPA2 family of Na/H
antiporters (NHAs). Supporting these hypotheses are the observations that amiloride and amiloride-based compounds inhibit 1) fluid secretion in isolated Malpighian tubules of dipterans (27, 32, 56) and 2) electrogenic K⫹/H⫹ antiport in vesicles
derived from highly purified apical membranes of K⫹-secreting cells of the Manduca midgut epithelium (83). A recent
study on Aedes Malpighian tubules by Kang’ethe and colleagues (34) has proposed that the apical cation/H⫹ exchanger
is an ortholog of mammalian NHE8 (SLC9A8), whereas a
more recent study on Drosophila Malpighian tubules by Day
and colleagues (21) has proposed that the exchanger is an
ortholog of bacterial NHAs [i.e., the bacterial K⫹ efflux (Kef)
family].
Parallel to the studies by Kang’ethe et al. (34) and Day et al.
(21), the present study reports our efforts over the course of the
past 4 years to identify and characterize the apical cation/H⫹
exchanger in Malpighian tubules of Aedes. We focused on the
Aedes ortholog of NHE8 that in mammals resides in the apical
membrane of renal proximal tubules and normal rat kidney
cells (3, 28, 92), where it mediates Na/H exchange (92).
Indeed, we cloned a cDNA from Aedes Malpighian tubules that
is identical to AeNHE8, which was cloned from an Aedes
cDNA library by Kang’ethe and colleagues (34). We detected
AeNHE8 immunoreactivity in principal cells of the distal
secretory segments, where it localizes to an intracellular compartment basal to, but not in, the brush border. Furthermore, the
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Piermarini PM, Weihrauch D, Meyer H, Huss M, Beyenbach
KW. NHE8 is an intracellular cation/H⫹ exchanger in renal tubules of
the yellow fever mosquito Aedes aegypti. Am J Physiol Renal Physiol
296: F730 –F750, 2009. First published February 4, 2009;
doi:10.1152/ajprenal.90564.2008.—The goal of this study was to
identify and characterize the hypothesized apical cation/H⫹ exchanger
responsible for K⫹ and/or Na⫹ secretion in the renal (Malpighian)
tubules of the yellow fever mosquito Aedes aegypti. From Aedes
Malpighian tubules, we cloned “AeNHE8,” a full-length cDNA encoding an ortholog of mammalian Na⫹/H⫹ exchanger 8 (NHE8). The
expression of AeNHE8 transcripts is ubiquitous among mosquito
tissues and is not enriched in Malpighian tubules. Western blots of
Malpighian tubules suggest that AeNHE8 is expressed primarily as an
intracellular protein, which was confirmed by immunohistochemical
localizations in Malpighian tubules. AeNHE8 immunoreactivity is
expressed in principal cells of the secretory, distal segments, where it
localizes to a subapical compartment (e.g., vesicles or endosomes),
but not in the apical brush border. Furthermore, feeding mosquitoes a
blood meal or treating isolated tubules with dibutyryl-cAMP, both of
which stimulate a natriuresis by Malpighian tubules, do not influence
the intracellular localization of AeNHE8 in principal cells. When
expressed heterologously in Xenopus laevis oocytes, AeNHE8 mediates EIPA-sensitive Na/H exchange, in which Li⫹ partially and K⫹
poorly replace Na⫹. The expression of AeNHE8 in Xenopus oocytes
is associated with the development of a conductive pathway that
closely resembles the known endogenous nonselective cation conductances of Xenopus oocytes. In conclusion, AeNHE8 does not mediate
cation/H⫹ exchange in the apical membrane of Aedes Malpighian
tubules; it is more likely involved with an intracellular function.
F731
MOSQUITO NHE8
localization of AeNHE8 in principal cells was not influenced
during periods of enhanced fluid secretion by the Malpighian
tubules. When expressed heterologously in Xenopus laevis oocytes, AeNHE8 mediates EIPA-sensitive Na/H exchange, but not
K/H exchange. In addition, the expression of AeNHE8 stimulates
an endogenous nonselective cation conductance in Xenopus
oocytes. Thus our data provide evidence that AeNHE8 mediates cation/H⫹ exchange across an intracellular membrane, but
not across the apical membrane, of Malpighian tubules.
MATERIALS AND METHODS
Mosquitoes and Tissue Isolations
Isolation of Total RNA and Synthesis of cDNA
All steps were performed under RNase-free conditions and at room
temperature unless specified otherwise. For RNA extractions, select
tissues (see above) and whole animals were centrifuged at 5,000 g for
3 min. The Ringer solution was aspirated, and the tissues or intact
animals were homogenized in 1 ml of TRIzol reagent (Invitrogen,
Carlsbad, CA) using a sintered-glass homogenizer. To extract total
RNA, we used a phenol:chloroform phase separation with an isopropyl-alcohol precipitation (17). To minimize contamination by DNA,
the resulting RNA was treated with DNase I (DNA-free, Ambion,
Austin, TX) according to the manufacturer’s protocol.
For RT-PCR and rapid amplification of cDNA ends (RACE),
single-stranded cDNA was generated from 2 ␮g of total RNA, using
oligo (dT)20 primers (Qbiogene, Carlsbad, CA) and the ThermoScript
RT-PCR System (Invitrogen). For relative mRNA expression, 0.4 ␮g
of total RNA was used to generate the cDNA.
Relative mRNA Expression
To compare relative levels of AeNHE8-transcript expression, we
followed previously published protocols that used a RT-PCR-based
approach to analyze the accumulation of PCR products during logarithmic amplification (78 – 80). In brief, cDNAs derived from the
following samples were used as templates for PCR: 1) 22 whole
female mosquitoes, 2) 110 Malpighian tubules pooled from 22 females, 3) guts (i.e., midgut and hindgut) pooled from 22 females, and
4) thorax and abdomens pooled from 22 females. The primer pairs
were designed to amplify 1) a 705-bp region of AeNHE8 (forward
primer: 5⬘-GGT GAC ACA AAT CAC GAT GC-3⬘, reverse primer:
5⬘-GG TAT TCG GAT CGC CTG GTA-3⬘); and 2) a 446-bp region
of Aedes ribosomal protein S3 (AeRbS3; forward primer: 5⬘-ATC
ATC ATC ATG GCC ACC CGT A -3⬘, reverse primer: 5⬘-CCA TTT
GGA TCC CAA GGC AAC A-3⬘). The RbS3 gene is known to be
expressed equally among insect tissues (78, 93). Thus it served as an
internal control for standardization.
The PCR protocol consisted of the following steps: 1) denaturation
for 4 min at 90°C with subsequent cooling to 72°C, 2) addition of 2.5
U of DNA Taq polymerase (Qbiogene), and 3) 40 amplification cycles
with an annealing temperature of 53°C for 30 s and an elongation
temperature of 72°C for 1 min. Starting at the end of the cycle 22, and
then for every other cycle afterward, the thermocycler was paused to
allow for removal of 8 ␮l of sample.
After completion of the PCR protocol, all of the removed samples
were separated by electrophoresis on a 1.5% agarose gel, which was
then stained with ethidium bromide and visualized with UV light. A
Fluor-S Multi-Imager (Bio-Rad, San Jose, CA) was used to digitize
the gel images, and Quantity One Software (Bio-Rad) was used to
quantify the optical densities of the resulting PCR products at cycle 36
(log phase) for AeNHE8 and cycle 24 (log phase) for AeRbS3. The
data are presented as the ratio of the optical density of the AeNHE8
PCR product to that of the AeRbS3 PCR product (i.e., the AeNHE8/
AeRbS3 ratio).
RT-PCR and RACE
The initial RT-PCR experiments on Aedes Malpighian tubule
cDNA were conducted before publication of the Aedes genome (45).
For this reason, we used the sequence of a putative NHE (GenBank
accession no. XM_307859) from the genome of the malaria mosquito
Anopheles gambiae to design the first set of primers. The standard
PCR protocol consisted of the following steps: 1) denaturation for 4
min at 94°C with subsequent cooling to 72°C, 2) addition of 2.5 U of
DNA Taq polymerase (Qbiogene), 3) 35 amplification cycles with an
optimized annealing temperature (50 – 60°C) and an elongation time
of 1 min/kb of the predicted PCR product.
Depending on the expected size of the PCR products, they were
separated on either 1 or 2% agarose gels by electrophoresis and
stained with ethidium bromide for detection under UV light. The PCR
products were 1) gel purified using a Qiaquick Gel Extraction Kit
(Qiagen, Valencia, CA), 2) ligated into a pGEM-T vector (Promega,
Mannheim, Germany), and 3) transformed into a competent Escherichia coli strain (DH5␣). After selection on LB-amp agar plates, E.
AJP-Renal Physiol • VOL
Antibodies
Charles River (Sulzfeld, Germany) was hired to raise and affinity
purify a polyclonal rabbit antibody against a synthetic peptide fragment of the predicted AeNHE8 protein. The peptide corresponded to
amino acid residues Ser511-Arg524 of the putative, cytosolic COOHterminal domain of AeNHE8. Before injection of the rabbits, the
peptide was covalently linked to keyhole-limpet hemocyanin. The
resulting anti-sera were affinity-purified with the synthetic peptide,
which was covalently linked to a HYDRA column (Squarix, Marl,
Germany).
A polyclonal guinea pig antibody (C23) raised against the Bsubunit of the V-type H⫹-ATPase from Manduca was kindly provided by the laboratory of Helmut Wieczorek (University of Osnabrück, Osnabrück, Germany). A monoclonal mouse antibody (␣5)
raised against the ␣-subunit of avian Na-K-ATPase developed by Dr.
Douglas Fambrough (John Hopkins University) was obtained from
the Developmental Studies Hybridoma Bank developed under the
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The mosquito colony was maintained as described by Pannabecker
and colleagues (52). Tissues were dissected from adult females (3–7
days after eclosion) that were anesthetized on ice and then decapitated. Malpighian tubules and the digestive tract were isolated in
Ringer solution as described by Pannabecker and colleagues (52). The
Ringer solution contained (in mM) 150 NaCl, 3.4 KCl, 1.7 CaCl2, 1.8
NaHCO3, 1.0 MgSO4, 5 glucose, and 25 HEPES, pH 7.1.
The five Malpighian tubules were transferred to a 1.5-ml microcentrifuge tube (USA Scientific, Ocala, FL) containing 0.5 ml of
Ringer solution on ice. The remaining gut tissue (i.e., midgut and
hindgut) and the carcass (i.e., thorax and abdomen) were transferred
to separate 1.5-ml microcentrifuge tubes also containing 0.5 ml of
Ringer solution on ice. The dissections were performed in the time
period of ⬃1 h, when at least 30 female mosquitoes were processed.
For whole-animal samples, anesthetized, intact mosquitoes were
transferred to a 1.5-ml tube on ice.
coli colonies containing the insert were cultured overnight in 5 ml of
LB-amp liquid media at 37°C. The plasmids were isolated (Qiaprep
Spin Miniprep Kit, Qiagen) and submitted for automated sequencing
(Sequence Laboratories, Göttingen, Germany) using T7 and Sp6
primers (Promega).
After obtaining the partial sequence of the NHE amplified from
Aedes Malpighian tubules, we amplified the 5⬘ and 3⬘ ends of the NHE
cDNA using a FirstChoice RLM-RACE Kit (Ambion). Combining the
sequencing data from the respective RACEs and the initial RT-PCR
allowed us to assemble a full-length cDNA, which was then 1) amplified
via PCR, 2) sequenced in both the 5⬘ and 3⬘ directions, and 3) submitted
into GenBank (accession no. EU760347). From here, we refer to this
cDNA, and the protein it encodes, as AeNHE8.
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MOSQUITO NHE8
auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of
Biological Sciences (Iowa City, IA). Both the C23 and ␣5 antibodies
recognize their respective antigens in Aedes Malpighian tubules (55,
81). In addition, a monoclonal mouse antibody (JL-8) raised against
recombinant full-length Aequorea victoria green fluorescent protein
was obtained from Clontech (Mountain View, CA).
Malpighian Tubule Western Blotting
Malpighian Tubule Immunohistochemistry
For immunohistochemistry, 150 tubules (from 30 female mosquitoes) were isolated in Ringer solution as described above and then
fixed in 1 ml of an ice-cold fixative solution containing 3% paraformaldehyde, 0.05% glutaraldehyde, and 0.05% picric acid in a 0.1 M
Na-phosphate buffer, pH 7.3. In some cases, 50 isolated tubules (from
10 female mosquitoes) were incubated for 30 min at room temperature
with 1 ml of Ringer solution containing 10⫺3 M dibutyryl-cAMP
(db-cAMP), or 1 ml of Ringer alone, before they were fixed.
For blood-feeding experiments, 50 tubules were isolated and fixed
from 10 female mosquitoes that were fed a blood meal. These
mosquitoes were placed individually in a 200-ml glass beaker covered
with a mesh netting and allowed to feed on the investigator’s arm until
repletion, i.e., between stages 4 and 5 on the Pilitt-Jones scale (58).
After mosquitoes had fed to repletion, they were allowed 5 min to
process the blood meal before they were anesthetized on ice and their
tubules were isolated as described above. Fifty tubules from 10 female
mosquitoes that were handled as above, but not offered a blood meal,
served as controls.
All of the above tubules were 1) fixed overnight at 4°C,
2) washed overnight twice in 70% ethanol at 4°C to remove the fixative,
and 3) submitted to the Clinical Sciences Histology Laboratory (Cornell University, Ithaca, NY) for routine paraffin embedding and
sectioning. The tubule sections (4-␮m thick) were adhered to ProbeOn
Plus glass slides (Fisher Scientific, Hampton, NH).
All immunohistochemical staining procedures were conducted at
room temperature in the Immunopathology Research and Development Laboratory (Cornell University) unless noted otherwise. The
tubule sections were 1) dewaxed with xylene; 2) rehydrated with an
ethanol series (100, 95, and 70% ethanol); and 3) treated with 0.5%
H2O2 in methanol for 10 min to inhibit endogenous peroxidase
activity. The sections were then rinsed briefly with 70% ethanol and
AJP-Renal Physiol • VOL
Generation of AeNHE8 cRNA for Expression in Xenopus Oocytes
A cDNA containing the entire open reading frame (ORF) of
AeNHE8 was subcloned into the pGH19 Xenopus expression vector
(71) and then sequenced in both the 5⬘ and 3⬘ directions (Cornell DNA
Sequencing Center). The resulting AeNHE8-pGH19 cDNA was used
as a template to synthesize capped cRNA, encoding AeNHE8 protein,
with a T7 mMessage mMachine kit (Ambion) according to the
manufacturer’s protocol. The AeNHE8 cRNA was purified using an
RNeasy MinElute Cleanup Kit (Qiagen) and stored at ⫺80°C in
nuclease-free H2O (Integrated DNA Technologies, Coralville, IA).
The AeNHE8-pGH19 cDNA was also modified by subcloning the
ORF of enhanced green-fluorescent protein (eGFP), kindly provided
by the laboratory of Dr. Walter F. Boron (Case Western Reserve
University), to the 3⬘ end of the AeNHE8 ORF. A Quikchange
site-directed mutagenesis kit (Stratagene, La Jolla, CA) was then used
to delete the stop codon between the ORFs of AeNHE8 and eGFP,
resulting in one continuous ORF. The AeNHE8-eGFP-pGH19 cDNA
was used as a template to synthesize capped cRNA (as described
above) encoding an AeNHE8-eGFP fusion protein.
Heterologous Expression in Xenopus Oocytes
Stage V-VI oocytes were isolated from Xenopus as described by
Romero and colleagues (63), and the following day the oocytes were
injected with 28 nl of AeNHE8 cRNA (1.0 ng/nl), AeNHE8-eGFP
cRNA (1.0 ng/nl), or nuclease-free H2O. The injected oocytes were
cultured at 16°C in the wells of a Falcon six-well tissue culture plate
(Becton Dickson, Franklin Lakes, NJ) containing OR3 media (63) for
1–14 days before any experiments commenced.
Western blotting. To verify that Xenopus oocytes translate the
AeNHE8 and AeNHE8-eGFP cRNAs into protein, Western blots were
performed on membrane fractions of oocytes. All of the following
steps were performed at room temperature unless noted otherwise.
Seven days after injection of oocytes with 1) nuclease-free H2O, 2) 28
ng of AeNHE8 cRNA, or 3) 28 ng of AeNHE8-eGFP cRNA, 50
oocytes from each group were transferred to 1.5-ml microcentrifuge
tubes (USA Scientific) containing 1 ml of solution 1 (see Table 1 for
composition). After the oocytes were rinsed three times with solution
1, they were snap frozen in liquid nitrogen and stored at ⫺80°C. On
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Western blotting was performed as described previously (81). In
brief, the proteins of a crude tubule homogenate (derived from 200
Malpighian tubules of 40 female mosquitoes) were separated by
molecular mass on a denaturing 17% polyacrylamide gel using a Mini
Protean 3 electrophoresis chamber (Bio-Rad, Hercules, CA). The
separated proteins were transferred to a nitrocellulose membrane
(SERVA Electrophoresis, Heidelberg, Germany) using a semidry
apparatus (Bio-Rad).
To detect AeNHE8 immunoreactivity, the nitrocellulose membrane
was 1) blocked for 60 min in a Tris-buffered saline (TBSNT; 20 mM
Tris 䡠 HCl, 500 mM NaCl, 0.02% NaN3, 0.05% Tween 20, pH 7.5)
plus 3% fish gelatin; 2) incubated for 60 min with the AeNHE8
antibody (diluted 1:1,000 in TBSNT plus 1% fish gelatin); and
3) washed three times (5 min each) with TBSNT. The nitrocellulose
membrane was then 1) incubated for 60 min with a secondary
antibody, i.e., alkaline phosphatase-conjugated goat anti-rabbit IgG
(Sigma) diluted 1:10,000 in TBSNT plus 1% fish gelatin; and 2) washed
three times (5 min each) with TBSNT. To visualize binding of the
antibodies, colorimetric substrates of alkaline phosphatase, i.e., nitro
blue tetrazolium (NBT; Sigma) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP; Sigma), were applied to the membrane
according to the manufacturer’s protocol. When a protein band appeared, the membrane was rinsed with double-distilled H2O.
washed for 5 min in PBS consisting of the following (in mM): 145
NaCl, 3.2 NaH2PO4, and 7.2 Na2HPO4, pH 7.3.
To block nonspecific binding, the sections were treated for 20 min
with 10% normal goat serum (Zymed, San Francisco, CA) that was
supplemented with a 2⫻ casein solution (Vector Laboratories, Burlingame, CA). The blocking solution was then removed and replaced
immediately with 1) PBS supplemented with 1⫻ casein solution
(PBS-casein) as a negative control, 2) the anti-AeNHE8 antibody
(diluted 1:100 in PBS-casein), 3) the C23 antibody (diluted 1:200 in
PBS-casein), or 4) the ␣5 antibody (diluted 1:3 in PBS-casein).
Incubation of the sections with the antibodies occurred overnight at
4°C in a humidified chamber. The following day, the sections were
rinsed briefly three times with PBS and then washed in PBS for 5 min.
To detect binding of the primary antibodies, the sections were first
incubated for 20 min with a biotinylated secondary antibody diluted
1:50 in PBS-casein; i.e., goat anti-rabbit IgG for the anti-AeNHE8
antibody, goat anti-guinea pig IgG for the C23 antibody, and goat
anti-mouse IgG for the ␣5 antibody (all from Vector Laboratories).
The sections were then 1) rinsed and washed in PBS as described
above, 2) incubated for 20 min with a streptavidin/horseradish peroxidase (HRP) solution (Zymed), and 3) rinsed and washed in PBS
again as described above. To visualize binding of the antibodies, a
chromogenic substrate of HRP (3-amino-9-ethylcarbazole, AEC;
Zymed) was applied to the sections for 10 –15 min. After extensive
washing with tap water the sections were dipped in hematoxylin
(15–30 s) to counterstain the tissue and covered with a coverslip for
viewing on an AX70 compound microscope (Olympus, Melville, NY).
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MOSQUITO NHE8
Table 1. Composition (in mM) of oocyte recording solutions
Solution
1
NaCl
NMDG-Cl
KCl
LiCl
CsCl
RbCl
MgCl2
CaCl2
HEPES
96
9
1
0
0
0
1
1.8
5
2
0
105
1
0
0
0
1
1.8
5
3
0.96
104
1
0
0
0
1
1.8
5
4
5
6
7
8
9
10
0.96
9
1
95.04
0
0
1
1.8
5
0.96
9
96.04
0
0
0
1
1.8
5
9.6
95
1
0
0
0
1
1.8
5
9.6
9
1
86.4
0
0
1
1.8
5
9.6
9
87.4
0
0
0
1
1.8
5
9.6
9
1
0
86.4
0
1
1.8
5
9.6
9
1
0
0
86.4
1
1.8
5
The pH of all solutions was adjusted to 7.5 with the dominant cation’s hydroxide salt or NMDG-OH. The osmolality of all solutions was adjusted to 210 ⫾
5 mosmol/kgH2O by adding double-distilled H2O or mannitol.
AJP-Renal Physiol • VOL
Oocyte Electrophysiology
The solutions used in electrophysiology experiments are described
in Table 1. When required, 100 mM stock solutions of the inhibitors
GdCl3, EIPA, benzamil, amiloride, diphenylamine-2-carboxylate
(DPC), or heptanol (all from Sigma-Aldrich, St. Louis, MO) were
diluted to their final concentrations in solution 1. All stock solutions
of the inhibitors were prepared with dimethylsulfoxide, except for
GdCl3 which is soluble in double-distilled H2O. For all experiments,
the solutions were held in 250-ml glass Erlenmeyer flasks or 50-ml
polypropylene centrifuge tubes (Fisher Scientific) and delivered by
gravity to a RC-3Z oocyte chamber (Warner Instruments, Hamden,
CT) at a flow rate of 4 ml/min. All oocytes were initially placed in the
chamber under superfusion with solution 1. Solution changes were
made with a Rheodyne Teflon eight-way rotary valve (model 5012,
Rheodyne, Rohnert Park, CA).
Preliminary experiments indicated that the basic electrophysiological properties (e.g., intracellular pH, membrane potential, and membrane conductance), and their responses to lowering extracellular Na⫹
concentration ([Na⫹]o), of oocytes injected with AeNHE8 cRNA were
comparable to those injected with AeNHE8-eGFP cRNA. Thus
oocytes injected with 28 nl of AeNHE8-eGFP cRNA (1.0 ng/nl) were
primarily used in electrophysiological experiments, because this cRNA
allowed the rapid verification of heterologous expression using in vivo
fluorescence. Oocytes injected with 28 nl of nuclease-free H2O served as
controls. Both the AeNHE8-eGFP- and H2O-injected oocytes were used
for electrophysiology experiments 5–14 days after injection.
Intracellular recordings of pH and membrane potential. To measure intracellular pH (pHi), pH-sensitive and voltage-sensitive electrodes were fabricated from thin-walled borosilicate glass (part no.
30-0077, Harvard Apparatus, Holliston, MA) following the protocol
of Romero and colleagues (63). The voltage from the pHi electrode
was recorded with the high-impedance “channel A” of a Duo773
electrometer (World Precision Instruments, Sarasota, FL). The membrane voltage (Vm) was recorded with an OC-725C oocyte clamp
(Warner Instruments). Inputs from the pHi and Vm electrodes were
recorded digitally by a Digidata 1440A data acquisition system
(Molecular Devices, Sunnyvale, CA) and the Clampex module of
pCLAMP software (Version 10, Molecular Devices).
To obtain the voltage that is specific to pHi, the response of the Vm
electrode was subtracted from that of the pHi electrode (“pHi ⫺ Vm”)
within the Clampex module. Before an oocyte was impaled with
microelectrodes, the pHi electrode was calibrated in the oocyte bath
using two pH standards (6.0 and 8.0, Fisher Scientific) and solution 1
(pH 7.50). Applying a linear regression to the resulting calibration
curve allowed the pHi ⫺ Vm values recorded in oocytes to be
converted into actual pHi measurements, using the Clampfit module of
the pCLAMP software (Version 10, Molecular Devices).
Two-electrode voltage clamping. Whole-cell currents of oocytes
were acquired with the OC-725 oocyte clamp (Warner Instruments).
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the day of analysis, the oocytes were thawed on ice in 300 ␮l of an
ice-cold homogenization buffer consisting of solution 1 supplemented
with Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL) and EDTA (5 mM). The oocytes were then homogenized in
their respective microcentrifuge tubes with a plastic pestle (Kontes,
Vineland, NJ), and the volume of the homogenate was adjusted to ⬃1
ml with ice-cold homogenization buffer.
To clear the homogenates of cell debris, they were centrifuged at
3,000 g for 10 min at 4°C. The resulting supernatants were transferred
to fresh 1.5-ml centrifuge tubes (Beckman, Fullerton, CA) and centrifuged in an OptimaMax ultracentrifuge (Beckman) at 100,000 g for
60 min at 4°C. After the supernatants were aspirated, the pellets
containing the membrane protein fractions were resuspended in 50 ␮l
of ice-cold homogenization buffer and measured for total protein
using a bicinchoninic acid protein assay (Pierce Biotechnology). To
the remaining volumes of the resuspended membrane fractions, an
appropriate volume of a 5⫻ Laemmli sample buffer (37) was added
and the samples were denatured at 100°C for 5 min.
Approximately 25 ␮g of membrane protein from each sample was
separated by molecular mass on a denaturing 8% polyacrylamide gel
using a XCell Surelock Mini-cell electrophoresis unit (Invitrogen).
The separated proteins were then transferred to an Immunoblot polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using an XCell II
Blot Module (Invitrogen).
To detect AeNHE8 or GFP immunoreactivity, the PVDF membrane
was 1) washed three times (5 min each) with Tween-Tris-buffered
saline (TTBS; 10 mM Tris 䡠 HCl, 150 mM NaCl, 0.01% Tween 20, pH
7.4); 2) blocked for 1 h with 5% nonfat dry milk dissolved in TTBS
(blocking buffer); and 3) incubated overnight at 4°C with the antiAeNHE8 antibody (diluted 1:100 in blocking buffer) or the anti-GFP
antibody (diluted 1:2,500 in blocking buffer). On the following day,
the PVDF membrane was 1) washed three times (5 min each) with
TTBS; 2) incubated for 1.5 h with a secondary antibody, i.e., HRPconjugated goat anti-rabbit IgG (Pierce Biotechnology) or goat antimouse IgG (Pierce Biotechnology) diluted 1:25,000 in blocking buffer;
and 3) washed three times (5 min each) with TTBS. To visualize
binding of the antibodies, a luminescent substrate of HRP (SuperSignal
West Pico, Pierce Biotechnology) was applied to the PVDF membrane according to the manufacturer’s protocol, and the luminescent
signal was detected with X-ray film (Pierce Biotechnology).
In vivo fluorescence. To visualize expression of AeNHE8-eGFP
protein in Xenopus oocytes, in vivo fluorescence was performed as
follows. One to 14 days after oocytes were injected with either 28 nl
of AeNHE8-eGFP cRNA (1.0 ng/nl) or nuclease-free H2O, the
oocytes were transferred to the wells of a Falcon 12-well tissue culture
plate (Becton Dickson) containing 1–2 ml of solution 1. The oocytes
were examined in the Cornell Microscopy and Imaging Facility with
an OV100 (Olympus) fluorescence-imaging system equipped with a
150-W xenon lamp, a 460- to 490-nm excitation filter, and a 510- to
550-nm emission filter.
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The data were recorded digitally by a MiniDigi-1A data acquisition
system (Molecular Devices) and AxoScope software (Version 10,
Molecular Devices). Both the membrane current (Im) and Vm microelectrodes were filled with 3 M KCl. For all Im recordings, the Vm was
clamped to a hyperpolarizing holding potential of 30 mV. For example, if the spontaneous Vm of the oocyte was ⫺40 mV, then the clamp
voltage was set at ⫺70 mV.
All current-voltage (I-V) relationships of oocytes were acquired and
analyzed following a previously-published protocol (57). In brief, the
oocytes were held at a Vm close to their spontaneous Vm in solution 1
before initiating the voltage-clamp protocol, which was controlled by the
Clampex module of the pCLAMP software (Molecular Devices).
Statistics
Transmembrane segments and topological prediction. To
construct a hypothetical membrane topology of the predicted
AeNHE8 protein, we compared its amino acid sequence and
hydrophobicity profile with those of two mammalian isoforms
for which experimentally derived topology maps exist, i.e.,
NHE1 and NHE3 (49, 75, 94). Although the predicted
AeNHE8 protein shares only ⬃25% amino acid sequence
identity with mammalian NHE1 and NHE3 (sequence alignment not shown), Fig. 1A shows that their hydropathic plots are
similar, especially within the NH2-terminal domain that contains the transmembrane (TM) segments.
As shown in Fig. 1B, we predict that AeNHE8 consists of 1) a
large NH2-terminal domain composed of 12 TM segments and
a reentrant loop that are connected by several endofacial and
exofacial loops, some of which are predicted to be glycosylated
or phosphorylated; and 2) a cytosolic COOH-terminal domain
with several predicted phosphorylation sites. The amino acid
residues that compose the putative TM segments are identified
in the alignment shown in Fig. 2, which also includes the
RESULTS
cDNA Cloning of AeNHE8
We cloned an NHE-like cDNA from Aedes Malpighian
tubules that is nearly identical to that of AeNHE8, the ortholog
of mammalian NHE8 cloned from an Aedes cDNA library by
Kang’ethe and colleagues (34). The AeNHE8 cDNA that we
cloned consists of 2,940 bp with a predicted open-reading
frame of 2,004 bp. As shown by the red bar in Supplemental
Fig. 1 (all supplemental material for this article are available on
the journal web site), the 5⬘-untranslated region (UTR) of
“our” AeNHE8 cDNA (accession no. EU760347) begins 76 bp
upstream of the cDNA cloned by Kang’ethe and colleagues
(accession no. AY326255). The additional 76 bp are contiguous with that of the Aedes genomic data but contain two
nucleotide substitutions not found in the genome (lower-case,
underlined letters in line 1 of Supplemental Fig. 1). Compared
with the cDNA cloned by Kang’ethe and colleagues, we find a
single nucleotide substitution of an A for a G in the 5⬘untranslated region (UTR; the * in line 1 of Supplemental Fig.
1). An A in this position is consistent with the genomic data.
The only difference within the ORF is a single silent substitution of an A in our AeNHE8 cDNA instead of a G (the * in line
9 of Supplemental Fig. 1). Thus the proteins encoded by both
cDNAs are identical. Although the A in our AeNHE8 cDNA is
inconsistent with the genomic data, this substitution may represent a polymorphism that is unique to the line of Aedes used
by our laboratory, because the same substitution was found
after a repetition of the RT-PCR for AeNHE8 on Malpighian
tubule cDNA derived from an independent generation of mosquitoes (data not shown). The 3⬘-UTR of our AeNHE8 cDNA
is identical to the one cloned by Kang’ethe and colleagues
(data not shown).
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Fig. 1. Hydropathic analysis and predicted topology of AeNHE8. A: aligned
hydropathic plots of AeNHE8 (red), human NHE1 (blue), and human NHE3
(green) generated in the BioEdit Sequence Alignment Editor software, version
7 (30) using the Kyte-Doolittle algorithm (36) with a window size of 15.
Breaks in the red and green lines represent gaps introduced by the sequence
alignment. Numbers indicate predicted transmembrane segments, and R indicates a predicted reentrant loop. B: predicted topology map of AeNHE8 based
on hydropathy plot in A. Transmembrane segments are numbered at their
emerging ends. Putative posttranslational modifications and regulatory sites are
also indicated (see text and Fig. 2 for details). The symbols are placed next to
one another if they share a putative phosphorylation site.
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Statistical tests were performed using Graphpad Prism 4 (Graphpad
Software, San Diego, CA). Comparisons between two groups (e.g.,
H2O-injected vs. AeNHE8 oocytes) were evaluated with unpaired
t-tests, whereas comparisons within AeNHE8 oocytes were assessed
with a one-way repeated-measures ANOVA and a Bonferroni posttest. The posttest was only used to compare mean values in AeNHE8
oocytes if the means were both 1) significantly different from those of
their respective H2O-injected oocytes, and 2) in the same direction
(i.e., positive or negative). To compare and categorize the mean
inward Im produced by various cations in AeNHE8 oocytes, a one-way
unpaired ANOVA with a Newman-Keuls posttest was used. To
determine whether an inhibitor had a significant effect, a one-sample
t-test was used with a hypothetical value of “0.0.” The mean percent
inhibitions produced by each inhibitor were compared by a one-way
unpaired ANOVA and categorized with a Newman-Keuls posttest.
Bioinformatic Analyses of AeNHE8 Protein
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amino acid sequences of NHE8 orthologs from Drosophila
(DrNHE8) and H. sapiens (HsNHE8).
Our topological model for AeNHE8 (Figs. 1B and 2) is
similar to that proposed by Kang’ethe and colleagues (34), but
with a few important modifications to accommodate the similar
hydrophobicity profiles (and amino acid sequences) shared
between AeNHE8 and the mammalian NHEs (i.e., NHE1 and
NHE3) in the predicted TM segments. Namely, we hypothesize
that residues 386-406 form a reentrant loop between TM
segments 9 and 10, instead of forming TM segment 10. In
addition, we propose a TM segment at residues 453-474 (i.e.,
TM segment 11) that is not predicted by the previous model.
Conserved and predicted features. The amino acid sequence
of AeNHE8 contains several regions that are highly conserved
among NHEs. In TM segments 4 and 9, AeNHE8 contains
residues (red text in Fig. 2) that are associated with sensitivity
to amiloride and its analogs (11, 49). In addition, AeNHE8
contains 1) residues in TM segment 4 (green text in Fig. 2) that
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Fig. 2. Annotated sequence alignment. The
amino acid sequence of AeNHE8, predicted by
its cDNA (accession no. EU760347), is aligned
with those of NHE8 from Drosophila melanogaster (DrNHE8; accession no. AAD32689)
and Homo sapiens (HsNHE8; accession no.
NP_056081) by the ClustalW algorithm (39).
The residue shading was performed by BioEdit
Sequence Alignment software, version 7 (30)
with a threshold of 100%. Identical residues are
shaded in black; similar residues are highlighted
in gray. Numbered horizontal bars indicate the
predicted transmembrane segments, and the dotted line indicates the reentrant loop, which are
depicted in Fig. 1B. Red text indicates residues
associated with sensitivity to amiloride and its
analogs. Green text indicates the residues conserved among all NHEs. Blue text indicates the
regions of high homology. See text for details.
Symbols are as in Fig. 1B.
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Expression of AeNHE8 Transcripts in Adult
Female Mosquitoes
To determine whether AeNHE8 transcripts are enriched in a
particular tissue of adult females, we used a RT-PCR-based
approach to estimate the expression of AeNHE8 relative to that
of Aedes ribosomal protein S3 (AeRbS3), which served as an
internal control. In insects, RbS3 transcripts are expressed to a
similar degree among a wide variety of tissues (78, 93).
Expression of AeNHE8 was measured in the following samples: 1) whole female mosquitoes, 2) Malpighian tubules,
3) gut (i.e., midgut and hindgut), and 4) thorax and abdomen.
As shown in Fig. 3A, expression of AeNHE8 is weak, but
detectable, in all of the assays between cycles 34 and 40 of the
PCR. In contrast, expression of the internal control (AeRbS3)
was robust and detectable in all of the assays as early as cycle
22 of the PCR.
Figure 3B shows the ratios of AeNHE8 to AeRbS3 expression (AeNHE8/AeRbS3 ratios) measured at the cycles indicated
by the boxes. If the AeNHE8/AeRbS3 ratio is greater than that
in the whole animal (dashed line in Fig. 3B), then AeNHE8
expression is enriched. Similarly, if the AeNHE8/AeRbS3 ratio
is lower than that in the whole animal, then AeNHE8 expression is reduced. Expression of AeNHE8 in the Malpighian
tubules is slightly below the average expression in the whole
animal, whereas expression in the gut and in the thorax and
abdomen are slightly above (Fig. 3B). In general, the AeNHE8/
AeRbS3 ratios show that 1) expression of AeNHE8 transcripts
is ubiquitous in the female mosquito, and 2) expression levels
of AeNHE8 are similar among the tissues examined.
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Fig. 3. Relative mRNA expression of AeNHE8. A: images of RT-PCR products for AeNHE8 and ribosomal protein AeRbS3 separated by agarose gel
electrophoresis. Every other PCR cycle is indicated, starting at cycle 22. The
boxed areas represent those bands selected for relative expression analysis.
B: ratios of AeNHE8 expression at cycle 36 to AeRbS3 expression at cycle 24.
Dashed line indicates the threshold ratio for enriched or reduced expression
relative to the whole animal.
Western Blotting of AeNHE8 in Malpighian Tubules
As indicated by the arrow in Fig. 4, the AeNHE8 antibody
detects a single, discrete band of protein in crude extracts of
Aedes Malpighian tubules. The immunoreactive band has a
molecular mass of ⬃67 kDa, which is slightly lower than the
size of the AeNHE8 monomer predicted by its cDNA (i.e.,
⬃74 kDa).
Immunolocalization of AeNHE8 in Malpighian Tubules
As shown in Fig. 5A, immunoreactivity for AeNHE8 occurs
exclusively in principal cells of Aedes Malpighian tubules.
Stellate cells show no evidence for staining. The discrete,
punctate immunostaining for AeNHE8 primarily localizes to a
subapical region of principal cells beneath the brush border
(arrows in Fig. 5A). In some principal cells, AeNHE8 immunolabeling occurs in other intracellular regions as well (e.g.,
Fig. 6A and see Fig. 8). In contrast to AeNHE8, immunoreactivity for the B subunit of the V-type H⫹-ATPase primarily
labels the brush border (arrowheads in Fig. 5B) and is occasionally found in intracellular compartments (e.g., Fig. 6B).
Thus our immunohistochemical data clearly indicate that
AeNHE8 is expressed primarily in intracellular compartments
of principal cells. No staining was detected when normal rabbit
serum or PBS was used in place of the AeNHE8 or B subunit
antibodies (data not shown).
To examine the distribution of AeNHE8 immunoreactivity in
relation to that of the V-type H⫹-ATPase and the Na-K-ATPase,
we labeled two consecutive sections (4 ␮m apart) of Malpighian
tubules: one section was incubated with the AeNHE8 antibody,
and the other was incubated with an antibody raised against 1) the
B subunit of the V-type H⫹-ATPase or 2) the ␣-subunit of the
Na-K-ATPase. It is known that the B subunit is expressed by
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are highly conserved among all eukaryotic NHEs (11); and
2) the regions of high homology in TM segments 6 and 7 (blue
text in Fig. 2), which are considered crucial for translocation of
Na⫹ and H⫹ (51, 76).
With regard to potential posttranslational modifications and
regulatory sites, Figs. 1B and 2 highlight the locations of three
putative N-glycosylation sites and several phosphorylation
sites as predicted by an in silico PROSCAN analysis (19).
Glycosylations and phosphorylations of NHEs are common
modifications associated with the maturation and acute regulation of the functional protein (reviewed by Refs 10, 23, 49,
51, 76). Note that the COOH-terminal domain of AeNHE8,
which is considered the regulatory domain of most NHEs,
contains three regions with predicted phosphorylation sites
(i.e., Ser548-Glu558; Pro580-Phe587; Leu601-Gln607) that are
highly conserved with HsNHE8. These regions are absent in
DrNHE8 (Fig. 2).
An analysis of the amino acid sequence of AeNHE8 with
SignalP 3.0 (4) predicts that the first 32 amino acid residues
(i.e., Met1-Ser32) form a signal-anchor peptide, as indicated by
the stars in Figs. 1B and 2. Signal anchors are involved with the
subcellular targeting, membrane insertion, and membrane anchoring of TM proteins (41, 73). In DrNHE8, a predicted
signal-peptide cleavage site occurs in a similar location (27).
Thus, in contrast to AeNHE8, the first TM segment of DrNHE8
may exist as a separate entity from the rest of the molecule, as
occurs for NHE3 of mammals (94). No consensus signalanchor or signal-peptide sequences are found in HsNHE8.
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both control (Fig. 8, A and C) and stimulated (Fig. 8, B and D)
Malpighian tubules. Staining for AeNHE8 occurs in subapical
and/or intracellular compartments, but is not found in the
luminal brush border (Fig. 8).
Heterologous Expression of AeNHE8 in Xenopus Oocytes
principal cells of both the distal and proximal segments of Aedes
Malpighian tubules, whereas the ␣-subunit is expressed only by
principal cells of proximal segments (55).
The AeNHE8 immunolabeling occurred in principal cells
that showed brush border (and sometimes intracellular) staining for the B subunit of the V-type H⫹-ATPase as shown by
the representative AeNHE8-positive principal cell in Fig. 6A.
Not all principal cells express AeNHE8 immunoreactivity,
regardless of their staining for the B subunit, even in principal
cells adjacent to those expressing AeNHE8 (Fig. 6A).
The principal cells that express AeNHE8 immunoreactivity
show an absence of detectable immunolabeling for the ␣-subunit
of the Na-K-ATPase (Fig. 7, A and B). We also observe the
corollary: i.e., principal cells expressing basolateral immunoreactivity for the ␣-subunit of the Na-K-ATPase show an absence of
detectable immunolabeling for AeNHE8 (Fig. 7, C and D).
To determine whether the immunolabeling for AeNHE8
redistributes to the brush border during diuresis, we examined
the localization of AeNHE8 in 1) Malpighian tubules of bloodfed mosquitoes, 5 min after they consumed a blood meal; and
2) isolated Malpighian tubules that were treated with the
known secretagogue db-cAMP (10⫺3 M) for 30 min. As shown
in Fig. 8, the immunolabeling for AeNHE8 is comparable in
Fig. 5. Immunoperoxidase localizations of
AeNHE8 and the B subunit of the V-type
H⫹-ATPase in female Malpighian tubules.
Representative localizations are shown of
AeNHE8 (A) and the B subunit of the V-type
H⫹-ATPase (B) in isolated Malpighian tubules of adult Aedes females. Red staining
identified by the arrows in A and arrowheads
in B represents labeling by the respective
antibodies. Sections are counterstained with
hematoxylin to stain nuclei and provide
contrast.
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Fig. 4. Expression of AeNHE8 immunoreactivity in female Malpighian tubules. Western blotting on crude homogenates from isolated Malpighian
tubules of adult Aedes females is shown. Arrow indicates the protein band
displaying AeNHE8 immunoreactivity. Molecular mass markers (in kDa) are
indicated.
To verify the heterologous expression of the AeNHE8 protein and the AeNHE8-eGFP fusion protein in Xenopus oocytes,
we conducted Western blotting on isolated membrane fractions
from oocytes 7 days after injection with H2O, AeNHE8 cRNA
(28 ng), or AeNHE8-eGFP cRNA (28 ng). As shown in strips
1–3 of Fig. 9A, the AeNHE8 antibody detects three discrete
protein bands (arrows a, b, and c) in membrane fractions from
the AeNHE8 and AeNHE8-eGFP oocytes that are not found in
those from the H2O-injected oocytes. In addition, a large band
of diffuse immunoreactivity is just visible around 250 kDa in
the AeNHE8 and AeNHE8-eGFP oocytes (bracket d in strips 2
and 3, Fig. 9A), but not in the H2O-injected oocytes (strip 1,
Fig. 9A). This large band is more apparent when the X-ray film
is overexposed to the chemiluminescent signal emitted from
the PVDF membrane (Supplemental Fig. 2). The finding of
multiple and diffuse immunoreactive bands in the AeNHE8 and
AeNHE8-eGFP oocytes is similar to results of Western blots
for mammalian NHE8 when it is overexpressed in COS-7 and
yeast cells (28, 29, 43).
As shown in strips 3 and 4 of Fig. 9A and Supplemental Fig.
2, the nearly identical patterns of immunoreactivity detected by
the AeNHE8 (strip 3) and GFP (strip 4) antibodies in the
AeNHE8-eGFP oocytes verifies 1) the specificity of the
AeNHE8 antibody and 2) that the unique bands (relative to
H2O-injected oocytes) are associated with the heterologous
expression of AeNHE8 protein. One exception is a single immunoreactive band around 55 kDa that is labeled by the AeNHE8
antibody (strips 1–3, Fig. 9A), but is not labeled by the GFP
antibody (strip 4, Fig. 9A). This band may represent a crossreaction between the AeNHE8 antibody and an endogenous membrane protein of Xenopus oocytes that shares an epitope with the
AeNHE8 peptide used to immunize the rabbits.
In AeNHE8 oocytes (strip 2 of Fig. 9A), bands a and b have
molecular masses of 92 and 97 kDa, respectively. Both are
greater than 1) the expected size of the AeNHE8 monomer (i.e.,
⬃74 kDa) and 2) the size of AeNHE8 immunoreactivity
detected in Malpighian tubules (Fig. 4). If bands a and b indeed
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Fig. 6. Immunoreactivity for AeNHE8 and
the V-type H⫹-ATPase in consecutive sections of female Malpighian tubules. Localizations are shown of AeNHE8 (A) and subunit B of the V-type H⫹-ATPase (B) in
isolated Malpighian tubules of adult Aedes
females. A and B: consecutive sections (4
␮m apart) of the same Malpighian tubule.
Red staining represents labeling by the respective antibodies. Sections are counterstained with hematoxylin to stain nuclei and
provide contrast. Note that AeNHE8 is not
present in every principal cell.
Thus these fractions of AeNHE8 protein and AeNHE8-eGFP
fusion protein appear to migrate anomalously of their mass.
To visualize expression of the AeNHE8-eGFP fusion protein
in Xenopus oocytes, we used in vivo fluorescence. Figure 9B
shows in vivo fluorescence images of oocytes 6 days after
injection with either H2O or AeNHE8-eGFP cRNA (AeNHE8eGFP). Whereas no fluorescence is detectable in the H2Oinjected oocyte, a robust fluorescent signal is observed over the
entire surface of the AeNHE8-GFP oocyte (Fig. 9B). This
fluorescent signal is detectable in AeNHE8-eGFP oocytes as
early as 2 days after injection and remains detectable for at
least 14 –17 days after injection (data not shown).
Taken together, the data from the Western blotting and
fluorescent imaging indicate that Xenopus oocytes heterologously express AeNHE8 and AeNHE8-eGFP proteins. Moreover, the data show that AeNHE8 is expressed as a protein of
Fig. 7. Immunoreactivity for AeNHE8 and
the Na-K-ATPase in consecutive sections of
female Malpighian tubules. Localizations are
shown of AeNHE8 (A and C) and the ␣-subunit of the Na-K-ATPase (B and D) in isolated
Malpighian tubules of adult Aedes females.
Consecutive sections (4 ␮m apart) of 1 tubule
are shown, respectively, in A, B, and C, D. Red
staining represents labeling by the respective
antibodies. Sections are counterstained with
hematoxylin to stain nuclei and provide contrast. Only principal cells of the proximal
segments of Aedes Malpighian tubules express basolateral immunoreactivity for the
Na-K-ATPase (55). Thus the AeNHE8-positive principal cells in A are from the distal
Malpighian tubule, and the AeNHE8-negative
principal cells in C are from the proximal
Malpighian tubule.
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correspond to AeNHE8, we would expect comparable bands to
appear at molecular masses that are ⬃30 kDa higher in
AeNHE8-eGFP oocytes, reflecting the extra mass added by the
fusion to eGFP. As seen in strip 3 of Fig. 9A, we observe such
bands (i.e., a and b) at molecular masses of 132 and 140 kDa,
respectively. These bands are also observed in AeNHE8-eGFP
oocytes with the GFP antibody (strip 4 of Fig. 9A).
In AeNHE8 oocytes, another band of immunoreactive protein
appears at a molecular mass of ⬃150 kDa (c in strip 2, Fig. 9A).
A similarly sized band is also detected in the AeNHE8-eGFP
oocytes with the AeNHE8 and GFP antibodies (c in strips 3 and
4 of Fig. 9A). Similarly, the large band of diffuse immunoreactivity in AeNHE8 oocytes (d in strip 2 of Fig. 9A and Supplemental Fig. 2) spans a similar range of molecular masses to that
detected by the AeNHE8 and GFP antibodies in AeNHE8-eGFP
oocytes (d in strips 3 and 4 of Fig. 9A and Supplemental Fig. 2).
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higher molecular mass than expected at the surface of Xenopus
oocytes. In contrast, Aedes Malpighian tubules express
AeNHE8 as a protein of lower molecular mass than expected
in an intracellular compartment of principal cells (Figs. 4
and 5A). We explain the reasons for these differences in the
DISCUSSION.
Electrophysiological Characterization of AeNHE8 in
Xenopus Oocytes
Basic properties of AeNHE8-expressing and H2O-injected
oocytes. As shown in Table 2, oocytes injected with cRNA
encoding AeNHE8-eGFP (hereafter referred to as AeNHE8
Fig. 9. Immunochemical and in vivo fluorescent detection of heterologously expressed
AeNHE8 in Xenopus oocytes. A: Western blotting on isolated membrane fractions from Xenopus oocytes 7 days after injection with H2O,
AeNHE8 cRNA (28 ng), or AeNHE8-enhanced green fluorescent protein (eGFP)
cRNA (28 ng). Labeled arrows and brackets
indicate protein bands displaying AeNHE8 or
GFP immunoreactivity not observed in H2Oinjected oocytes. No GFP immunoreactivity
was observed in membrane fractions from
H2O-injected or AeNHE8 oocytes (data not
shown). Molecular mass markers (in kDa) and
antibodies used for each strip are indicated.
Strip numbers are referred to in the text. See
Supplemental Fig. 2 for overexposed versions
of the same blots. B: fluorescent images of
Xenopus oocytes injected with H2O or 28 ng
of AeNHE8-eGFP cRNA. Dashed circle outlines the location of the H2O-injected oocyte,
of which only the less-pigmented vegetal pole
is visible.
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Fig. 8. Immunoreactivity for AeNHE8 in
control and stimulated female Malpighian tubules. Representative localizations are shown
of AeNHE8 in Malpighian tubules of adult
Aedes females. A: tubule isolated from an
unfed mosquito (control). B: tubule isolated
from a blood-fed mosquito, 5 min after ending a blood meal (stimulated). C: isolated
Malpighian tubule incubated in Ringer solution for 30 min (control). D: isolated Malpighian tubule incubated in 10⫺3 M dibutyryl
(db)-cAMP for 30 min (stimulated). Red
staining represents labeling by the AeNHE8
antibody. Sections are counterstained with
hematoxylin to stain nuclei and provide contrast. Note that AeNHE8 is not present in
every principal cell (e.g., A and C).
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Table 2. Properties of AeNHE8-expressing and H2O-injected
oocytes in solution 1
Oocytes
pHi
Vm, mV
Table 3. Parameters of current-voltage relationships in
AeNHE8-expressing and H2O-injected oocytes
gm, ␮S
AeNHE8
7.35⫾0.02* (n⫽29) ⫺34.1⫾1.4* (n⫽31) 1.64⫾0.15* (n⫽36)
H2O-injected 7.19⫾0.02 (n⫽25) ⫺52.1⫾1.7 (n⫽17) 0.35⫾0.02 (n⫽18)
Values are means ⫾ SE. pHi, intracellular pH; Vm, membrane voltage; gm,
membrane conductance. Measurements of gm were obtained with 2-electrode
voltage clamping, where the oocyte is clamped at a holding potential that is 30
mV hyperpolarized relative to the spontaneous Vm. *Significant difference
from H2O-injected oocytes (P ⬍ 0.0001).
Fig. 10. Current-voltage (I-V) plots of AeNHE8-expressing and H2O-injected
oocytes. Negative membrane current (Im) values represent the net movement of
positive charge into or negative charge out of the cell (inward current), whereas
positive Im values represent the net movement of positive charge out of or
negative charge into the cell (outward current). Data were acquired in solution
1. Values are means ⫾ SE based on the number of oocytes in parentheses.
Missing error bars indicate values too small to draw.
AJP-Renal Physiol • VOL
Slope Conductance
(␮S) Between ⫺120
and ⫺160 mV
Erev, mV
AeNHE8 (n ⫽ 18)
H2O-injected (n ⫽ 15)
2.44⫾0.32*
1.04⫾0.08
9.47⫾1.22*
0.72⫾0.06
⫺28.3⫾1.4*
⫺41.3⫾1.7
Values are means ⫾ SE. Erev, reversal potential. *Significant difference
from H2O-injected oocytes (P ⬍ 0.0001).
to dissect the transport events that affect the pHi of AeNHE8
oocytes from those that influence the electrical properties
(e.g., Vm).
Effects of Na⫹ and EIPA on pHi and Vm. A typical experiment is shown in Fig. 11A. The AeNHE8 oocyte is initially
superfused with a solution containing 96 mM Na⫹ (solution 1).
After the switch to a solution in which NMDG⫹ completely
replaces Na⫹ (solution 2, Table 1), pHi begins on a declining
slope (a in Fig. 11A), presumably due to AeNHE8 operating in
“reverse” mode, i.e., H⫹ uptake and Na⫹ extrusion. In addition, the Na⫹ replacement causes an initial sharp hyperpolarization of Vm by ⬃25 mV (A in Fig. 11A), followed by a more
gradual hyperpolarization and stabilization of Vm, consistent
with the presence of a conductive pathway. Restoring the
normal [Na⫹]o results in 1) a reversal of the pHi trajectory and
a gradual recovery of pHi to its resting level (b in Fig. 11A),
and 2) a sharp repolarization of Vm (B in Fig. 11A).
Repeating the removal of [Na⫹]o repeats the effects on pHi
and Vm (c and C in Fig. 11A). However, restoring the normal
[Na⫹]o in the presence of 0.1 mM EIPA arrests the pHi
recovery (d in Fig. 11A). In contrast, the Vm fully repolarizes in
the presence of EIPA (D in Fig. 11A). Thus EIPA inhibits the
Na⫹-dependent recovery of pHi, but it does not block the
conductive pathway. Subsequent washout of EIPA has no
effect on Vm (E in Fig. 11A), but starts a gradual recovery of
pHi (e in Fig. 11A). In the oocyte shown, the recovery of pHi
after EIPA washout is nominal, indicating that the effects of
the inhibitor are not easily reversed. However, in other oocytes
a more noticeable recovery ensued after washout of EIPA, as
indicated by the data summary in Fig. 11D.
Repeating the above protocol on a H2O-injected oocyte
causes minor effects on pHi (Fig. 11B) relative to those
observed in the AeNHE8 oocyte. The solution changes result in
similar directional changes to Vm as seen in the AeNHE8
oocyte, but with lower magnitudes (Fig. 11B). However, in the
H2O-injected oocyte, restoring the normal [Na⫹]o in the presence of 0.1 mM EIPA causes a slow depolarization of Vm that
is reversible upon the washout of EIPA (D in Fig. 11B).
Figure 11, C–E, summarizes the ⌬pHi/⌬t and ⌬Vm measurements in AeNHE8 (shaded bars) and H2O-injected (open bars)
oocytes. The specific step changes in [Na⫹]o and EIPA are
depicted in Fig. 11C. The ⌬pHi/⌬t of AeNHE8 oocytes is
significantly greater in magnitude than that of the H2O-injected
oocytes after all solution changes, except after restoration of
[Na⫹]o in the presence of EIPA, in which case the rates are
equal (Fig. 11D). In AeNHE8 oocytes, the rate of pHi recovery
during the EIPA washout is significantly lower than during the
restoration of normal [Na⫹]o without EIPA (Fig. 11D), indicating that on average the EIPA treatment has residual, but not
irreversible, effects on AeNHE8 activity.
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oocytes) have 1) a high pHi, 2) a depolarized Vm, and 3) a
greater membrane conductance compared with oocytes injected with H2O. The higher pHi of AeNHE8 oocytes is
consistent with the activity of an NHE expected to operate in
“forward” mode, i.e., Na⫹ uptake in exchange for H⫹ extrusion in solution 1. In contrast, a depolarized Vm and markedly
enhanced membrane conductance of AeNHE8 oocytes are not
expected from the activity of an electroneutral NHE.
I-V relationships further illustrate the presence of the unexpected conductive pathway in AeNHE8 oocytes. As shown in
Fig. 10, the I-V plot of AeNHE8 oocytes is curvilinear, increasing in voltage dependence with hyperpolarizing Vm. In contrast, the I-V plot of the H2O-injected oocytes is relatively
linear and shallow throughout the range of voltages examined.
At hyperpolarizing Vm, the membrane conductance is 13 times
greater in AeNHE8 oocytes than in H2O-injected oocytes
(Table 3). The reversal potential (Erev) of the AeNHE8 oocytes
is significantly more positive by ⬃10 mV compared with the
H2O-injected oocytes (Table 3).
In the following sections, we characterize the electrophysiological properties of the AeNHE8 oocytes in more detail using
experimental manipulations of the bathing solution. Our aim is
Oocytes
Slope Conductance
(␮S) Between ⫹20
and ⫺60 mV
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The hyperpolarization associated with removing [Na⫹]o
(Fig. 11E) and the depolarizations associated with restoring
normal [Na⫹]o, without or with EIPA, are significantly greater
in AeNHE8 oocytes compared with H2O-injected oocytes (Fig.
11E), documenting the activation of a new membrane conductance in AeNHE8 oocytes. Again, the new membrane conductance in AeNHE8 oocytes is not affected by EIPA (Fig.
11E), in contrast to the profound effect of EIPA on ⌬pHi/⌬t
(Fig. 11D).
Effect of monovalent cation substitutions on pHi and Vm. In
Fig. 12A, an AeNHE8 oocyte is initially superfused with a
AJP-Renal Physiol • VOL
solution containing 96 mM Na⫹ (solution 1). Switching to a
solution in which the [Na⫹]o is lowered to 1 mM (solution 3)
results in similar effects on pHi and Vm (a and A in Fig. 12A)
as observed for removal of [Na⫹]o in Fig. 11A. Again, the
effects are reversible upon restoration of normal [Na⫹]o (b and
B in Fig. 12A).
Repeating the step reduction of [Na⫹]o leads again to the
gradual cellular acidification and the sharp depolarization of
Vm (c and C in Fig. 12A). However, in the switch to a solution
in which Li⫹ substitutes for the lowered Na⫹ (solution 4, Table
1), the recovery of pHi is markedly blunted (d in Fig. 12A).
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Fig. 11. Effects of removing and restoring extracellular Na⫹ concentration ([Na⫹]o) in the absence or presence of EIPA on intracellular pH (pHi) and Vm.
A: representative recordings of pHi and membrane voltage (Vm) in an AeNHE8 oocyte. Extracellular concentrations (in mM) of Na⫹ and EIPA are indicated above
the recordings. When Na⫹ is removed, it is replaced by NMDG⫹. Solution changes are indicated by dashed vertical lines. In the pHi tracing, the dashed lines
(labeled a, b, c, etc.) are slopes to indicate the rates of pHi change (⌬pHi/⌬t). A time bar is included. B: representative recordings of pHi and Vm in a H2O-injected
oocyte, using a protocol similar to that in A. C: extracellular solution changes (arrows) after which the ⌬pHi/⌬t and ⌬Vm measurements were made in AeNHE8
and H2O-injected oocytes. The step-changes in [Na⫹]o associated with the solution changes are indicated (values in mM). D: summary of ⌬pHi/⌬t measurements.
Shaded bars represent ⌬pHi/⌬t values of AeNHE8 oocytes (number of oocytes in parentheses) after the solution changes depicted in C. The value for removing
[Na⫹]o represents the combined mean ⌬pHi/⌬t from periods a and c in A. The open bars represent H2O-injected oocytes at similar periods. E: summary of ⌬Vm
measurements. Shaded bars represent ⌬Vm values of AeNHE8 oocytes measured 90 s after the solution changes depicted in C. The value for removing Na⫹
represents the combined mean ⌬Vm from A and C in A. The open bars represent H2O-injected oocytes after similar solution changes. Values are means ⫾ SE.
A missing error bar indicates a value too small to draw. Curved brackets connecting shaded and open bars represent unpaired t-tests that resulted in significant
differences. Solid horizontal lines between shaded bars represent Bonferroni post hoc comparisons from a 1-way paired ANOVA. *P ⬍ 0.05, **P ⬍ 0.01,
***P ⬍ 0.001.
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MOSQUITO NHE8
Accordingly, AeNHE8 oocytes accept Li⫹ for exchange transport with H⫹, but not as effectively as Na⫹. In contrast, the Li⫹
solution repolarizes the Vm of the oocyte (D in Fig. 12A) as
effectively as the Na⫹ solution (B in Fig. 12A), suggesting
similar membrane conductances for Li⫹ and Na⫹. Again, we
observe that pHi and Vm respond independently to experimental maneuvers.
Switching to a solution in which K⫹ replaces Na⫹ (solution
5) does not reverse the acidification of the oocyte (f in Fig.
AJP-Renal Physiol • VOL
12A). Instead, the cellular acidification that began upon lowering [Na⫹]o (e in Fig. 12A) continues unperturbed. In contrast,
the Vm strongly depolarizes in the high-K⫹ solution (F in Fig.
12A), which reflects a membrane conductance far greater for
K⫹ than Na⫹ and Li⫹ (B and D in Fig. 12A). Thereafter,
replacing the extracellular K⫹ with Na⫹ results in 1) a profound reversal of the pHi trajectory followed by a robust
recovery of pHi (g in Fig. 12A) and 2) a partial repolarization
of Vm to about ⫺22 mV (G in Fig. 12A). We assume that the
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Fig. 12. Effects of replacing extracellular Na⫹ with Li⫹ or K⫹ on pHi and Vm. A: representative recordings of pHi and Vm in an AeNHE8 oocyte. Extracellular
concentrations (in mM) of Na⫹, Li⫹, and K⫹ are indicated above the recordings. When Na⫹ is lowered, it is replaced by NMDG⫹, if not by Li⫹ or K⫹. Solution
changes are indicated by dashed vertical lines. In the pHi tracing, the dashed lines (labeled a, b, c, etc.) are slopes to indicate the rates of pHi change (⌬pHi/⌬t).
A time bar is included. B: representative recordings of pHi and Vm in a H2O-injected oocyte, using a protocol similar to that in A. C: extracellular solution changes
(arrows) after which the ⌬pHi/⌬t and ⌬Vm measurements were made in AeNHE8 and H2O-injected oocytes. The step-changes in extracellular cation
concentration ([cation]o) associated with the solution changes are indicated (values in mM). D: summary of ⌬pHi/⌬t measurements. Shaded bars represent
⌬pHi/⌬t values of AeNHE8 oocytes (number of oocytes in parentheses) after the solution changes depicted in C. The value for lowering [Na⫹]o represents the
combined mean ⌬pHi/⌬t from periods a and c in A. The value for restoring [Na⫹]o represents the combined mean ⌬pHi/⌬t from periods b and g in A. The open
bars represent H2O-injected oocytes at similar periods. E: summary of ⌬Vm measurements. Shaded bars represent ⌬Vm values of AeNHE8 oocytes measured 90 s
after the solution changes depicted in C. The value for lowering [Na⫹]o represents the combined mean ⌬Vm from periods A and C in A. The open bars represent
H2O-injected oocytes after similar solution changes. Values are means ⫾ SE. Curved brackets connecting shaded and open bars represent unpaired t-tests that
resulted in significant differences. Solid horizontal lines between shaded bars represent Bonferroni post hoc comparisons from a 1-way paired ANOVA. **P ⬍
0.01, ***P ⬍ 0.001.
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AJP-Renal Physiol • VOL
Repeating the above protocol on a H2O-injected oocyte
results in 1) a low inward Im after clamping of the oocyte
(double-headed arrow in Fig. 13B); and 2) similar directional
changes to Im associated with the solution changes as seen in
the AeNHE8 oocyte, but with much lower magnitudes (Fig.
13B). Notably, the high-K⫹ solution does not produce a large
inward Im in H2O-injected oocytes (g in Fig. 13B) compared
with that observed in the AeNHE8 oocytes (g in Fig. 13A).
The protocol in Fig. 13A was used to also examine the
effects of adding Cs⫹ and Rb⫹ in place of Na⫹. A typical
experiment is not shown. Instead, Fig. 13C summarizes the
inward Im produced by each of the monovalent cations normalized to the inward Im produced by restoring normal [Na⫹]o.
The inward Li⫹ current is significantly lower (⬃40%) than the
inward Na⫹ current, whereas the inward Cs⫹ current is similar
to the inward Na⫹ current. The inward K⫹ and Rb⫹ currents
are significantly greater (⬃75%) than the inward Na⫹ current.
Inhibitor sensitivity of Im. In Fig. 14A, an AeNHE8 oocyte is
initially superfused with a solution containing 96 mM Na⫹
(solution 1), and then the oocyte is clamped at a holding
potential that is 30 mV hyperpolarized to the spontaneous Vm
(double-headed arrow in Fig. 14A), resulting in an inward Im.
Switching to a solution in which [Na⫹]o is lowered to 10 mM
(solution 6) causes the inward Im to approach zero and stabilize
(a in Fig. 14A). Restoring the normal [Na⫹]o restores the
inward Im (b in Fig. 14A).
Repeating the step reduction of [Na⫹]o repeats the effects on
Im (c in Fig. 14A). However, restoring the normal [Na⫹]o in the
presence of 0.1 mM Gd3⫹ reduces the inward Im by more than
half (d in Fig. 14A). Accordingly, Gd3⫹ blocks the conductive
influx of Na⫹. Washing out Gd3⫹ slowly restores the inward Im
(e in Fig. 14A).
Repeating the above protocol on a H2O-injected oocyte (Fig.
14B) results in 1) a low inward Im after of clamping the oocyte
(double-headed arrows in Fig. 14B); 2) similar directional
changes to Im as seen in the AeNHE8 oocyte associated with
lowering and restoring [Na⫹]o, but with lower magnitudes
(e.g., a and b in Fig. 14B); and 3) no inhibitory effect on the
small inward Im associated with restoring normal [Na⫹]o in the
presence of Gd3⫹ (d in Fig. 14B).
The protocol in Fig. 14 was used to also examine the effects of
other inhibitors on the inward Na⫹ current, including benzamil,
EIPA, amiloride, DPC, and heptanol. Figure 14C summarizes the
percent inhibition of the inward Im (upon restoring normal
[Na⫹]o) by each of these compounds in AeNHE8 oocytes. As
shown in Fig. 14C, Gd3⫹, benzamil, EIPA, and amiloride significantly inhibit ⌬Im, whereas DPC and heptanol do not. The most
effective inhibitors are Gd3⫹ and benzamil, blocking 40 –50% of
the inward Im, followed by EIPA and amiloride, which block
⬃25% of the inward Im. In H2O-injected oocytes, none of the
compounds significantly inhibit the small inward Im values (data
not shown).
DISCUSSION
Cloning and Expression of the AeNHE8 cDNA
In the present study, we have cloned an ortholog of mammalian
NHE8 from the Malpighian tubules of the yellow-fever mosquito
Aedes aegypti. Besides a few minor differences in the 5⬘-UTR and
the open-reading frame (Supplemental Fig. 1), the cDNA and the
protein it encodes are identical to AeNHE8 cloned from an Aedes
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AeNHE8 oocyte does not immediately repolarize to its original
spontaneous Vm because it likely accumulated intracellular K⫹
in the previous superfusion with the high-K⫹ solution. Notably, this K⫹ loading apparently did not take place in the
H2O-injected oocyte (see below and G in Fig. 12B).
In Fig. 12B, we repeated the above protocol on a H2Oinjected oocyte, which elicits minor effects on pHi. The solution changes result in similar directional changes to Vm as seen
in the AeNHE8 oocyte, but with lower magnitudes, except
when Na⫹ is replaced with K⫹ (Fig. 12B). As in the AeNHE8
oocytes, high extracellular K⫹ concentration strongly depolarizes Vm (F in Fig. 12B), but the subsequent return to normal
[Na⫹]o does not leave H2O-injected oocytes loaded with K⫹
(G in Fig. 12B).
Figure 12, C–E, summarizes the ⌬pHi/⌬t and ⌬Vm measurements in AeNHE8 (shaded bars) and H2O-injected (open bars)
oocytes. The specific step changes in extracellular concentrations of Na⫹, Li⫹, and K⫹ are depicted in Fig. 12C. The
⌬pHi/⌬t of AeNHE8 oocytes is significantly greater in magnitude than that of the H2O-injected oocytes under all conditions
except when Li⫹ replaces Na⫹, in which case the rates are
similar (Fig. 12D). In the AeNHE8 oocytes, the replacement of
Na⫹ with K⫹ does not recover pHi; instead, oocytes continue
to acidify, albeit at a lower rate than when NMDG⫹ replaces
Na⫹ (Fig. 12D). Accordingly, Li⫹ can partially substitute for
Na⫹ in the maintenance of pHi, but K⫹ cannot.
The hyperpolarization of Vm associated with lowering
[Na⫹]o (Fig. 12E) and the depolarizations associated with
restoring the normal [Na⫹]o or adding extracellular Li⫹ (Fig.
12E) are significantly greater in AeNHE8 oocytes than in
H2O-injected oocytes. Upon the replacement of extracellular
Na⫹ with K⫹, the depolarization of Vm was similar in AeNHE8
and H2O-injected oocytes (Fig. 12E). These observations indicate that the new conductance in AeNHE8 oocytes is sensitive to monovalent cation concentrations of Na⫹, Li⫹, and K⫹.
Monovalent cation preference of Im. In view of the substantial voltage responses of AeNHE8 oocytes, together with their
increased membrane conductance, we explored the cation preference of the conductive pathway using two-electrode voltage
clamping. In Fig. 13A, an AeNHE8 oocyte is initially superfused with a solution containing 96 mM Na⫹ (solution 1), and
then the oocyte is clamped at a holding potential that is 30 mV
hyperpolarized to the spontaneous Vm (double-headed arrow in
Fig. 13A). The inward Im of ⬎100 nA corresponds to the
conductive influx of a cation or the conductive efflux of an
anion. Switching to a solution in which the [Na⫹]o is reduced
to 10 mM (solution 6) causes the Im to reverse from a
substantial inward to a small outward current (a in Fig. 13A).
Restoring the normal [Na⫹]o restores the inward Im (b in Fig.
13A). It follows that hyperpolarizing and depolarizing currents
are carried by Na⫹.
Repeating the step reduction of [Na⫹]o repeats the effect on
Im (c in Fig. 13A). However, switching to a solution in which
Li⫹ replaces Na⫹ (solution 7) results in a blunted inward Im (d
in Fig. 13A). Accordingly, the conductive pathway is less
permeable to Li⫹ than to Na⫹, which is confirmed by the
subsequent replacement of extracellular Li⫹ with Na⫹ (e in
Fig. 13A). Thereafter, lowering Na⫹ reduces the inward Im
again (f in Fig. 13A). Switching to a solution in which K⫹
replaces Na⫹ (solution 8) results in the largest inward Im (g in
Fig. 13A).
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MOSQUITO NHE8
cDNA library by Kang’ethe and colleagues (34). In view of the
similar hydrophobicity profiles and amino acid sequences shared
between AeNHE8 and the mammalian NHEs (i.e., NHE1 and
NHE3) in the predicted TM segments (Fig. 1A), we hypothesize
that AeNHE8 has 12 TM segments and one reentrant loop (Figs.
1B and 2). Our predicted topological model for the AeNHE8
protein is consistent with that proposed recently for NHE isoforms
from a wide variety of eukaryotic organisms (see supplemental
figures in Ref. 11).
Our RT-PCR-based approach to measure AeNHE8-mRNA
expression indicates that AeNHE8 transcripts are expressed ubiquitously in the female mosquito. That is, the AeNHE8 mRNA is
present at a low level in all samples examined (detectable by PCR
cycles 34 – 40, Fig. 3A) and does not appear to be substantially
AJP-Renal Physiol • VOL
enriched in transporting epithelia, such as the Malpighian tubules
and gut, relative to its expression in the whole animal (Fig. 3B). In
other insects, such as Drosophila (21, 27) and the tobacco hornworm Manduca (Weihrauch D, unpublished observations), expression of NHE8 transcripts is also ubiquitous and not enriched
within a particular tissue. Among vertebrates, ubiquitous expression of NHE8 transcripts is common (29, 43, 87, 88), but in
mammals, NHE8 transcripts appear to be enriched in the
kidneys and skeletal muscle (29, 43).
Expression of AeNHE8 Protein in Aedes Malpighian Tubules
Although Western blotting for AeNHE8 in Aedes Malpighian tubules indicates that the protein is expressed at a
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Fig. 13. Effects of monovalent cations on Im of AeNHE8 oocytes. A: representative recording of Im in an AeNHE8 oocyte. Negative Im values represent
the movement of positive charge into the cell or negative charge out of the cell (inward current). The double-headed arrow indicates the start of the voltage
clamp. The Vm of the oocyte and extracellular concentrations of cations (in mM) of Na⫹, Li⫹, and K⫹ are indicated above the recording. When Na⫹ is
lowered, it is replaced by NMDG⫹ if not by Li⫹ or K⫹. Solution changes are indicated by dashed vertical lines. A time bar is included. B: representative
recording of Im in a H2O-injected oocyte, using a protocol similar to that in A. C: summary of relative effects of monovalent cations on inward currents
of AeNHE8 oocytes. The shaded bars represent the inward current produced by replacing the lowered [Na⫹]o with various monovalent cations using the
protocol in A. All Im values are standardized to the inward Im produced upon restoration of the normal [Na⫹]o (i.e., Na⫹ and the dashed line). Respective
currents from H2O-injected oocytes have been subtracted. Values are means ⫾ SE, based on measurements from the number of AeNHE8 oocytes in
parentheses. Lower-case letters indicate grouping of means by Newman-Keuls post hoc comparisons from a 1-way unpaired ANOVA.
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MOSQUITO NHE8
molecular mass that is ⬃10 kDa lower than predicted by its
cDNA (Fig. 4), the lower mass is in good agreement with that
observed for AeNHE8 expressed heterologously in PS120
fibroblasts and yeast cells (34). Mammalian NHE8 also exhibits a lower than expected molecular mass (by ⬃10 kDa) when
expressed in intracellular compartments as an “immature”
incompletely glycosylated form (28, 29, 43). The “mature”
fully glycosylated form of mammalian NHE8 appears at molecular masses ⬃20 –30 kDa greater than expected on Western
blots and reaches the plasma membrane (28, 29). In parallel,
AeNHE8 in Malpighian tubules may be expressed in intracellular compartments as an immature form.
Immunohistochemical localization of AeNHE8 in Malpighian tubules verifies our interpretation of the Western blot.
AeNHE8 localizes primarily to a subapical compartment of
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principal cells (Fig. 5A). In adult female Aedes, the subapical
cytoplasm of principal cells is occupied by numerous membrane-bound concretions (6, 8). Using transmission electron
microscopy, we have also observed the presence of 1) Golgi
complexes, 2) vesicular-like structures (e.g., endosomes or
secretory vesicles), and 3) rough endoplasmic reticulum in the
subapical cytoplasm of principal cells (Piermarini PM, unpublished observations). Thus AeNHE8 may be expressed in one
or more of the aforementioned organelles.
Consecutive sections of immunolabeled Malpighian tubules
show that the AeNHE8-positive principal cells express the B
subunit of the V-type H⫹-ATPase (Fig. 6), but not the ␣-subunit of the Na-K-ATPase (Fig. 7). Thus AeNHE8 is most likely
expressed by principal cells of the distal, secretory segments of
the tubules, because the V-type H⫹-ATPase is found in prin-
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Fig. 14. Effects of inhibitors on Im of AeNHE8 oocytes. A: representative recording of Im in an AeNHE8 oocyte. In this tracing, the effects of Gd3⫹ on Im are
shown. Negative Im values represent the movement of positive charge into the cell or negative charge out of the cell (inward current). The double-headed arrow
indicates the start of the voltage clamp. The Vm of the oocyte and extracellular concentrations of Na⫹ and Gd3⫹ (in mM) are indicated above the recording. When
Na⫹ is lowered, it is replaced by NMDG⫹. Solution changes are indicated by dashed vertical lines. A time bar is included. B: representative recording of Im in
a H2O-injected oocyte, using a protocol similar to that in A. C: summary of the relative effects of inhibitors on the inward currents of AeNHE8 oocytes. The
shaded bars represent the percent inhibition of the inward Im associated with restoration of normal [Na⫹]o using the protocol in A. Concentrations of the inhibitors
are indicated. The inhibition is calculated by comparing the ⌬Im after restoration of normal [Na⫹]o in the absence of an inhibitor (e.g., b in A) to that after
restoration of normal [Na⫹]o in the presence of an inhibitor (e.g., d in A). Values are means ⫾ SE, based on measurements from the number of AeNHE8 oocytes
in parentheses. Lower-case letters indicate grouping of means by Newman-Keuls post-hoc comparisons from a 1-way unpaired ANOVA. Significant inhibition
of the inward Im by the inhibitor (**P ⬍ 0.01 and ***P ⬍ 0.001).
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Functional Characterization of AeNHE8 in Xenopus Oocytes
To characterize the function of AeNHE8 in Xenopus oocytes
using measurements of pHi and Vm, the protein must be
AJP-Renal Physiol • VOL
expressed in the plasma membrane. Our immunochemical,
in vivo fluorescent, and electrophysiological data in Xenopus
oocytes are all consistent with the heterologous expression of
AeNHE8 in the plasma membrane.
First, Western blotting in Xenopus oocytes indicates that the
AeNHE8 protein and AeNHE8-eGFP fusion protein are primarily expressed at molecular masses ⬃20 – 40 kDa greater
than predicted by their cDNAs (bands a and b in Fig. 9A). In
addition, we observe broad, diffuse bands of AeNHE8 (and
GFP) immunoreactivity (e.g., d in Fig. 9A and Supplemental
Fig. 2) that appear at even greater molecular masses than bands
a and b. These findings are consistent with the expression of
mature forms of AeNHE8 and AeNHE8-eGFP that reach the
plasma membrane, as observed for mammalian NHE8 (28, 29).
In contrast, Malpighian tubules express AeNHE8 as an immature form of lower molecular mass than expected within
intracellular compartments (Figs. 4 and 5A). Second, the fluorescence associated with the expression of an AeNHE8-eGFP
fusion protein is detectable over the entire surface of the oocyte
(Fig. 9B), documenting the expression of AeNHE8 in the
plasma membrane. Third, the display of unique physiological
activities in AeNHE8 oocytes compared with H2O-injected
counterparts confirms the overexpression of AeNHE8 in the
plasma membrane. Most significantly, AeNHE8 oocytes exhibit large changes in pHi and Vm in response to step changes
in extracellular cation concentrations. These changes are not
observed in the H2O-injected oocytes.
It is important to point out that previous studies have
documented endogenous NHE activity in Xenopus oocytes (12,
13, 33, 46, 64, 70). However, it is unlikely that this endogenous
oocyte NHE contributes to the robust NHE activity that we
observe in the AeNHE8-expressing oocytes, for the following
reasons.
First, isolating oocytes from Xenopus ovaries with a collagenase digestion markedly decreases the activity of the endogenous oocyte NHE (70). In the present study, all of the oocytes
were extracted from Xenopus ovaries with collagenase. Second, the activity of the endogenous oocyte NHE steadily
declines within 3 days after its isolation from the ovary (13,
70). In the present study, the pHi recordings in the oocytes
were conducted at least 6 days after their isolation. Third, the
proton extrusion activity of the endogenous oocyte NHE is
activated by a pronounced acidification of pHi (12, 14) or by
cell shrinkage (33). In the present study, the AeNHE8 oocytes
were evaluated near the resting pHi (typically ⬎7.2) and the
osmolalities of the experimental solutions were constant (Table
1). Fourth, although in one instance the heterologous expression of a membrane-bound protein, i.e., the Plasmodium falciparum chloroquine resistance transporter, was shown to enhance the activity of an endogenous oocyte NHE, removing
and restoring [Na⫹]o had no detectable effects on the resting
pHi of these oocytes (46). In contrast, lowering or restoring
[Na⫹]o in AeNHE8-expressing oocytes results in noticeable
effects on pHi within a few minutes of the solution change
(Figs. 11A and 12A). Taken together, we attribute the pHi
changes in the AeNHE8-expressing oocytes to the activity of
the AeNHE8 protein.
AeNHE8 activity. The Xenopus oocytes expressing AeNHE8
show changes in pHi that are consistent with the activity of an
EIPA-sensitive Na/H exchanger. First, AeNHE8 oocytes have
a higher pHi than H2O-injected oocytes (Table 2). Second,
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cipal cells of both the proximal and distal segments of Aedes
Malpighian tubules, whereas the Na-K-ATPase is only found
in principal cells of the proximal segments of Aedes Malpighian tubules (55). The presence of AeNHE8 immunoreactivity in one principal cell and not in the adjacent principal cell
of Aedes Malpighian tubules (e.g., Figs. 6 and 8) is consistent
with observations made in Malpighian tubules of Drosophila,
where principal cells express distinct genetic repertoires along
the axial length of the tubule (67).
In Aedes Malpighian tubules, the localization of AeNHE8 is
not influenced 5 min after mosquitoes finish a blood meal or 30
min after treatment of isolated tubules with db-cAMP (Fig. 8).
Thus the transporter is not trafficked from an intracellular pool
to the brush border during conditions of enhanced NaCl and
fluid secretion. In contrast, the immunoreactivity for ␤-actin
undergoes a dramatic redistribution from the cytosolic regions
to the brush border 5 min after Aedes mosquitoes consume a
blood meal and 15– 60 min after treatment of isolated Malpighian tubules with 10⫺3 or 10⫺4 M db-cAMP (35). The
redistribution of ␤-actin is hypothesized to participate in microvillar growth of the brush border, the trafficking of organelles to the brush border, and the assembly of the V-type
H⫹-ATPase holoenzyme in the brush border (35), all of which
are expected to promote transepithelial fluid secretion. Clearly,
AeNHE8 does not participate in this promotion of diuresis.
Our localization of AeNHE8 in subapical regions of Aedes
Malpighian tubules conflicts with that of Kang’ethe and colleagues (34), who report that AeNHE8 localizes to the apical
membrane of principal cells. It is unlikely that the conflict
arises from the different AeNHE8 antibodies used, because
both were raised against similar synthetic AeNHE8 peptides
(i.e., Ser511-Arg524 in our study, and Ser511-Leu525 for Kang’ethe
et al.). However, on close examination, the AeNHE8 immunoreactivity in Fig. 8 of Kang’ethe et al. does not occur throughout the brush border as expected for a protein localized to the
apical membrane, but is near the brush border and appears
punctate, similar to what we observed. Moreover, the images
of immunoreactivity taken in our study, which show greater
resolution than those of Kang’ethe and colleagues, reveal a
clear separation between AeNHE8 expressed in discrete subapical compartments and the B subunit of the V-type H⫹ATPase expressed in the apical brush-border membrane (Fig. 5).
The intracellular localization of an NHE8 isoform is not
unprecedented: 1) in Drosophila Malpighian tubules, immunolabeling for DrNHE8 localizes to intracellular compartments of
principal cells, but not to the plasma membrane (21); 2) human
NHE8 expressed in COS7 cells localizes to the mid- and
trans-Golgi networks (43); 3) NHE8 immunoreactivity is beneath the brush border in renal proximal tubules of 1-day-old
rats (3); and 4) the putative ortholog of NHE8 in C. elegans
(NHX-8) appears to localize to vesicular compartments of
seam cells (44). To date, the only examples in which a plasma
membrane localization of an NHE8 isoform has clearly been
demonstrated is in the brush border of renal proximal tubules
from 10-day-old and adult rats (3, 28) and in the apical
membrane of normal rat kidney (NRK) cell lines (92).
F747
MOSQUITO NHE8
AJP-Renal Physiol • VOL
Our data do not support a conductance due to the electrogenic 2Na⫹/H⫹ antiport by AeNHE8. First, restoring normal
[Na⫹]o in the presence of EIPA and/or amiloride 1) fully
blocks the pHi recovery of AeNHE8 oocytes, but it does not
affect the repolarization of Vm (Fig. 11); and 2) does not
effectively block the inward Im (Fig. 14). If AeNHE8 was an
electrogenic 2Na⫹/H⫹ exchanger, then EIPA and/or amiloride
would be expected to abolish the pHi recovery, repolarization
of Vm, and the inward Im, as in the case of 2Na⫹/H⫹ antiport
in crustaceans and echinoderms (1, 2, 66). Second, the conductive pathway of AeNHE8 oocytes favors the transport of
K⫹ over that of Na⫹ and Li⫹ (Figs. 12 and 13), whereas the
proton extrusion activity of AeNHE8 oocytes favors Na⫹ over
Li⫹ and K⫹ (Fig. 12). An electrogenic NHE would be expected
to display the same cation preferences for both the conductive
pathway and the proton transport. Third, Fig. 12 shows that Vm
and pHi change independently of each other in AeNHE8
oocytes, and preliminary results from our laboratory suggest
that clamping the membrane potential of AeNHE8 oocytes
does not influence their resting pHi (Piermarini PM, unpublished observations).
Thus our data tell of an independent conductive pathway that
is activated by the heterologous expression of AeNHE8. In
support of this conclusion, the relative ability of K⫹, Na⫹, and
Li⫹ to produce inward currents in AeNHE8-expressing oocytes
(Fig. 13C) is remarkably similar to that observed for various
endogenous nonselective cation conductances of frog oocytes,
such as those activated by membrane suction (i.e., stretch) or
by exposure to maitotoxin or DIDS (9, 22, 68, 91). Furthermore, the sequence of the monovalent cations carrying inward
Im (i.e., Rb⫹ ⬇ K⫹ ⬎ Cs⫹ ⬇ Na⫹ ⬎ Li⫹; Fig. 13C) suggests
a type III or IV Eisenman sequence (25), indicating that the
conducting pores consist of “weak” cation-binding sites that
favor dehydrated permeation of ions rather than hydrated
permeation. That is, the conductive pores favor larger cations
that shed their hydration shells most readily. The preference for
permeation of large cations with low hydration enthalpies is
comparable to that observed previously for endogenous nonselective cation conductances in Xenopus oocytes (22, 91).
Third, the most effective inhibitors of the inward Im associated
with returning normal [Na⫹]o in AeNHE8 oocytes are Gd3⫹
and benzamil (Fig. 14). Gadolinium is a well-known blocker of
endogenous nonselective cation conductances in Xenopus oocytes (9, 22, 61, 77, 90). Although the inhibition of the inward
Im by Gd3⫹ was not complete, nonselective cation conductances in oocytes are difficult to fully block by Gd3⫹ once
they have been activated (9, 61, 77). Furthermore, our
finding that benzamil is a more effective inhibitor than
amiloride is consistent with the presence of nonselective
cation channels (9, 38).
Although we cannot rule out that independent parts of the
AeNHE8 molecule mediate the pHi and voltage changes, we
prefer the hypothesis that AeNHE8 mediates the Na⫹-dependent pHi changes and a protein endogenous to the oocyte
mediates the voltage changes. The mechanisms that activate
the endogenous conductive protein are unclear, but one study
suggests that nonselective cation conductances of Xenopus
oocytes are mediated by TRPC1 mechanosenstive cation channels (40).
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removing [Na⫹]o results in an acidification of pHi and restoring
normal [Na⫹]o results in an alkalinization of pHi, which is
blocked by EIPA (Fig. 11, A and D). These observations in
oocytes agree with those made of AeNHE8 expressed in PS120
fibroblasts, where EIPA-sensitive uptake of 22Na⫹ is enhanced
(34). However, we find that in Xenopus oocytes the proton
extrusion activity of AeNHE8 has a preference for Na⫹ over
Li⫹ and K⫹ (Fig. 12, A and D), which is in conflict with the
activity of AeNHE8 expressed in yeast and characterized in
reconstituted proteoliposomes (34). In proteoliposomes, the
affinities of AeNHE8 for Na⫹ and K⫹ are comparable (34). A
similar conflict occurs when mammalian NHE8 is studied in
cells vs. proteoliposomes. For example, rat NHE8 mediates
Na/H exchange, but not K/H exchange, in the apical plasma
membranes of NRK cells that endogenously express the exchanger (92). In contrast, human NHE8 mediates both Na/H
and K/H exchange when expressed in yeast and characterized
in reconstituted proteoliposomes (43).
The above conflicts indicate that the physiological properties
of NHE8 isoforms depend on the expression system and
membranes in which they are characterized. In Xenopus oocytes (Fig. 9) and in NRK cells (92), NHE8 isoforms are
primarily expressed as mature forms that reach the plasma
membrane. However, in yeast, NHE8 isoforms are primarily
expressed as immature forms in intracellular compartments
(34, 43). Thus the characterization of NHE8 isoforms in
Xenopus oocytes and in NRK cells reflects an activity in the
plasma membrane, whereas the characterization of NHE8 in
yeast proteoliposomes reflects the activity of the transporter in
an intracellular compartment. A similar explanation has been
offered previously by Zhang and colleagues (92). We conclude
that AeNHE8 mediates EIPA-sensitive Na/H exchange in
plasma membranes. Still, it is possible that AeNHE8 permits
K/H exchange when expressed in the membranes of intracellular organelles, either in proteoliposomes of yeast or in the
subapical compartments of Aedes Malpighian tubules.
Conductive activity. The Xenopus oocytes expressing AeNHE8
in the present study also express a large membrane condutance,
as indicated by the following. First, the AeNHE8 oocytes have
a depolarized Vm and enhanced membrane conductance compared with H2O-injected oocytes (Table 2). Second, the I-V
plot of AeNHE8 oocytes reveals an increasing membrane
conductance at hyperpolarizing voltages that is not observed in
the H2O-injected oocytes (Fig. 10). Furthermore, Erev is significantly more positive in AeNHE8 oocytes than in the H2Oinjected oocytes (Table 3). Third, removing or lowering [Na⫹]o
prominently hyperpolarizes Vm (Fig. 11, A and E) and decreases the inward flow of current (Fig. 13A). Together, these
data indicate that AeNHE8 oocytes posses a conductive pathway in their plasma membranes, presumably for Na⫹, not
found in the H2O-injected oocytes. The pathway could arise
from 1) electrogenic exchange by AeNHE8 (e.g., 2Na⫹/H⫹
antiport), similar to that observed in membrane vesicles of
crustaceans and echinoderms (1, 2, 66); 2) an intrinsic cation
conductance within the AeNHE8 protein, as proposed for the
rat electroneutral Na-HCO3 cotransporter (16, 20); or 3) an
endogenous cation conductance of the oocyte that is activated
by heterologous expression of AeNHE8 protein, similar to the
conductances previously reported in Xenopus oocytes overexpressing heterologous membrane-bound proteins (46, 53,
72, 74).
F748
MOSQUITO NHE8
Is AeNHE8 an Apical Cation/H⫹ Exchanger in Aedes
Malpighian Tubules?
What Might Be the Role of Intracellular AeNHE8 in
Principal Cells?
We hypothesize that AeNHE8 participates in regulating the
pH of at least one type of organelle in principal cells, such as
the membrane-bound concretions, endosomes, secretory vesicles, and/or the Golgi network. The proper sorting, targeting,
and recycling of proteins and vesicles by organelles are highly
dependent on the pH maintained within the lumen of organelles
(42, 54). One mechanism by which the luminal pH of an
organelle is regulated at a particular steady state is through the
activity of a proton “leak” pathway, whose molecular identity
remains unknown (54). At least in mammals, the intracellular
NHEs (i.e., NHE6, NHE7, NHE8, and NHE9) are considered
likely candidates that may contribute to this proton leak pathway in the various organelles of the secretory and endocytotic
pathways (43, 50).
Given the prominent subapical localization of AeNHE8 in
principal cells, AeNHE8 is likely to contribute to the regulation
of pH in organelles that maintain and/or regulate transport
processes in the brush border of principal cells. For example, it
is possible that AeNHE8 is localized to endosomes or vesicles
involved with the trafficking and/or recycling of membranebound proteins to and from the brush border. Such processes
may be especially crucial to proteins in the apical membrane,
because the brush border of Aedes Malpighian tubules is
enriched with mitochondria that not only generate the ATP for
fueling the apical V-type H⫹-ATPase (86) but also are likely to
generate damaging free radicals via oxidative phosphorylation
(i.e., ATP generation). Thus AeNHE8 could play an important
role in a recycling pathway that maintains the integrity of
proteins and organelles located in the brush border. Alternatively, AeNHE8 could contribute toward the regulation of pH
in organelles (e.g., secretory vesicles) of principal cells that are
AJP-Renal Physiol • VOL
The Search for the Elusive Apical Cation/H⫹
Exchanger Continues
Compared with the combined 11 members of the CPA
superfamily in mammals (9 NHEs and 2 NHAs), only 5
members are found in insects (3 NHEs and 2 NHAs). Of these
five, orthologs of mammalian NHE3 (59) and NHE8 (the
present study) have been ruled out as potential apical cation/H⫹ exchangers in Malpighian tubules of adult female
Aedes, whereas the orthologs of mammalian NHE3, NHE6,
and NHE8 have been ruled out in Malpighian tubules of
Drosophila (21).
In Drosophila, transcripts encoding the two putative NHA
proteins (orthologs of the bacterial K⫹ efflux, Kef, family) are
enriched in Malpighian tubules, and both of these proteins
localize to the brush border of principal cells (21). Furthermore, overexpression of these genes in Malpighian tubules
increases levels of K⫹ and/or Na⫹ in the secreted fluid (21). To
date, NHAs have not been studied in Aedes, but in larvae of the
malarial mosquito Anopheles gambiae, NHA1 immunoreactivity is found in Malpighian tubules (48, 62). Thus the current
data from Drosophila and Anopheles suggest that NHAs are
the best candidates for apical cation/H⫹ exchangers in Malpighian tubules of Aedes. Making NHAs even more attractive is
their potential for mediating K⫹/2H⫹ or Na⫹/2H⫹ exchange (62),
which would enable the exchanger to be driven by the high
apical Vm of principal cells that is established by the V-type
H⫹-ATPase. A stoichiometry of K⫹(Na⫹)/nH⫹ is thermodynamically feasible for exporting K⫹ and Na⫹ into the tubule
lumen.
ACKNOWLEDGMENTS
We thank the laboratories of Drs. William A. Horne, Huai-hu Chuang, and
Robert E. Oswald at the Cornell University College of Veterinary Medicine for
generously supplying the oocytes used in this study. We are also indebted to
the laboratory of Dean Michael I. Kotlikoff (Cornell University College of
Veterinary Medicine) for providing equipment used in molecular biology and
Western blotting. Last, we thank Patricia Fisher, Kenneth Lau, and Mary Lou
Norman (all of Cornell University) for excellent technical assistance.
GRANTS
Financial support from the following sources made this work possible: K01
DK-080194-01 awarded to P. M. Piermarini from the National Institute of
Diabetes and Digestive and Kidney Diseases; SFB 431 awarded to Markus
Huss from the Deutsche Forschungsgemeinschaft (DFG); WE 2868/1-2 from
the DFG and a Discovery grant from the Natural Sciences and Engineering
Research Council of Canada awarded to D. Weihrauch; IBN 0078058 awarded
to K. W. Beyenbach from the National Science Foundation. The sabbatical
support of K. W. Beyenbach with a Mercator Visiting Professorship from the
DFG is gratefully acknowledged.
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