Phylogeny and cloning of ion transporters in mosquitoes
Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA
* Author for correspondence (e-mail: sarjeet.gill{at}ucr.edu)
Accepted 18 July 2003
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Summary |
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Key words: V-ATPase, membrane transport, ion regulation, proton-motive force, sodium/proton exchanger, mosquito, cation-coupled chloride cotransporter
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Introduction |
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Ion regulation is governed by the concerted actions of several membrane
transport proteins, intracellular signaling molecules and brain-derived
factors that regulate these proteins. Unlike vertebrates, in which ion
homeostasis can be achieved by ultrafiltration, insects operate at low blood
pressure and thus require hormonal signaling from the brain to regulate active
ion transport. Brain-derived factors or hormones provide a means of regulating
transporter activities. Several peptides that control diuresis and
antidiuresis have been identified in insect Malpighian tubules
(Coast, 1996). Signaling
molecules involved in these opposing actions of the peptides also differ, in
that cAMP stimulates fluid secretion and cGMP promotes antidiuresis.
Natriuretic peptides that stimulate secretion by Malpighian tubules have
been isolated from mosquitoes (Petzel et
al., 1987). However, the molecular targets of these peptides in
stimulating ion and fluid transport can only be gleaned from studies that use
pharmacological agents that act on known targets. Of special interest are
processes affected by application of bafilomycin A1, ouabain,
bumetanide and amiloride, which respectively inhibit V-type
H+-ATPase, Na+/K+-ATPase,
Na+/K+/Cl--cotransporters (CCCs) and
sodium/proton exchangers (NHEs).
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Models for ion transport in insects |
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The V-ATPase is a large oligomeric complex consisting of two distinct
sectors: the membrane-bound V0 sector and the intracellular
V1 sector (Forgac,
1999; Nishi and Forgac,
2002
). Protons generated from cellular respiration appear to be
the major substrate for V-ATPase since mitochondrial poisoning with
dinitrophenol abolishes measurable V-ATPase activity and, subsequently, ion
transport. Furthermore, V-ATPases are present in close physical proximity to
mitochondria, enabling protons to be channeled efficiently through the proton
pump (also see Harvey and Wieczorek,
1997
). The role of V-ATPase in ion transport is further supported
by its inhibition by bafilomycin A1, which in parallel inhibits
transport in insect epithelia. Consistent with this role, immunohistochemical
analyses have demonstrated the presence of subunits of V-ATPase in
K+-transporting midgut membranes
(Klein, 1992
;
Sumner et al., 1995
;
Zhuang et al., 1999
) and
mosquito Malpighian tubules (Filippova et
al., 1998
; Weng et al.,
2003
; Pullikuth et al., submitted). Nearly all subunits of
V-ATPase have been cloned, and the expression patterns of some have been
examined (Filippova et al.,
1998
; Gill et al.,
1998
; Graf et al.,
1994a
,b
;
Merzendorfer et al., 2000
;
Pietrantonio and Gill, 1995
),
showing that they colocalize with the portasomes
(Zhuang et al., 1999
).
Structural analysis now implies that portasomes are actually the V1
sector of V-ATPase (Radermacher et al.,
1999
; Rizzo et al.,
2003
).
The V-ATPase transports H+ but not K+, yet
K+ is the only ion that crosses the entire midgut in lepidopterans
(Cioffi and Harvey, 1981).
Further biochemical characterization led to the hypothesis that the GCAM
contains a K+/H+ antiporter that uses the proton-motive
force generated by V-ATPase in GCAM to drive transport
(Harvey, 1992
;
Harvey et al., 1983a
;
Wieczorek et al., 1991
). This
transports K+ out to the lumen in an electrogenic exchange for
luminal protons (Azuma et al.,
1995
). Thus, current models for insect ion transport incorporate
V-ATPase in transporting cells as one of the major determinants of transport
that favors activation of secondary transport processes for ion and fluid
effluxes (Wieczorek, 1992
;
Wieczorek et al., 1991
,
1999
). However, the
Na+/K+-ATPase may also be involved in fluid and ion
transport, for example in insect Malpighian tubules (M. J. Patrick, K.
Aimanova, H. R. Sanders and S. S. Gill, submitted).
The lepidopteran apical proton pump is apparently electrogenic, exchanging
2H+ for every cation efflux
(Azuma et al., 1995;
Grinstein and Wieczorek, 1994
;
Lepier et al., 1994
). In
lepidopteran goblet membrane preparations, vesicle acidification by V-ATPase
was inhibited by bafilomycin A1, whereas outward-directed
K+ or Na+ transport was inhibited by amiloride and
harmaline; features that indicate involvement of a K+/H+
exchanger and Na+-transport mechanisms. V-ATPase could acidify
independently of extracellular K+ when K+ efflux was
blocked by amiloride, indicating that V-ATPase and K+-transporting
molecules are different (Wieczorek et al.,
1991
). Furthermore, antibodies that blocked V-ATPase activity and
ATP-dependent H+ transport had no effect on
K+/H+ exchange. Together, these results led to the
hypothesis that the lepidopteran K+/nH+
exchanger is involved in electrogenic K+ efflux aided by the
proton-motive force generated by the electrogenic V-ATPase
(Grinstein and Wieczorek,
1994
; Harvey and Wieczorek,
1997
; Wieczorek et al.,
1991
).
Despite the accepted role of V-ATPase in energizing epithelial transport,
little is known about the molecular identity of transporters that operate in
parallel with V-ATPase. However, pharmacological approaches with known
inhibitors of NHEs, such as amiloride and its related compounds, suggest that
a proton/cation exchanger is intricately involved in ion transport in mosquito
Malpighian tubules. In addition, involvement of other transporters, including
those similar to bumetanide-sensitive sodium/chloride cotransporters and
ouabain-sensitive Na+/K+-ATPase, contributes in varying
degrees to ion transport mechanisms in many insects (reviewed in
Pannabecker, 1995). Clearly,
molecular identification of these transporters is a necessary step in
conclusively assigning the involvement of such transporters to regulating ion
transport in mosquitoes.
Whole-tubule assays and short-circuit and other electrophysiological
analyses have helped to understand ion fluxes and thus fluid secretion in
Malpighian tubules (Beyenbach,
1995; Beyenbach et al.,
2000
). Acute activation of ion and fluid transport appears to be
correlated with cAMP-dependent mechanisms. Fluid secretion by isolated
Malpighian tubules can be stimulated by peritubular application of cAMP
analogues or by activating processes that sustain higher cellular cAMP such as
active adenylate cyclase or inhibition of phosphodiesterases
(Petzel et al., 1987
). Similar
properties could be mimicked by the application of mosquito natriuretic
peptides (MNPs). These brain-derived peptides possess diuretic properties, and
their release is possibly controlled by activation of stretch receptors
following ingestion of a blood meal
(Petzel et al., 1987
;
Fig. 1).
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Molecular characterization of ion transport proteins |
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Sodium/proton exchangers (NHEs)
Members of solute carrier family 9 are involved in exchanging protons for
cations (Na+ or K+), thereby regulating cellular and
systemic pH and ionic concentrations. In mammals, NHEs function in a variety
of cells and intracellular locations, indicating that members of this family
perform ubiquitous housekeeping functions as well as more specialized
physiological roles (Burckhardt et al.,
2002; Counillon and
Pouyssegur, 2000
; Orlowski and
Grinstein, 1997
). Cation transport dictated by the transmembrane
voltage established by apical V-ATPase in insect epithelium could be mediated
through members of the NHE family. Support for this notion comes from the
pharmacological properties of K+ and Na+ transport in
insect epithelia. Peritubular application of amiloride inhibits basal fluid
secretion in Malpighian tubules of Aedes aegypti
(Hegarty et al., 1991
), and
serotonin (5-HT) stimulates secretion in Rhodnius prolixus
(Maddrell and O'Donnell,
1992
). This effect probably does not involve basal membrane
Na+ channels, as no detectable effect on basal membrane voltages
was observed (Hegarty et al.,
1991
). Amiloride is also an Na+ channel antagonist,
which confounds the above issues. However, D. melanogaster Malpighian
tubules were shown not to express any known Na+ channels
(Giannakou and Dow, 2001
). The
amiloride derivative EIPA, which is more potent towards NHEs, does indeed
affect tubular pH and recovery from acid load, lending support to the
involvement of NHE-like molecules in apical membrane transport
(Petzel, 2000
).
Structure and function of mammalian NHEs
In mammals, eight distinct NHEs have been cloned. These exchangers exhibit
differential tissue distribution, cellular localization and transport
properties, indicating their specialized functions in particular tissues. A
detailed review of mammalian NHEs can be found in Orlowski and Grinstein
(1997), Counillon and
Pouyssegur (2000
) and
Burckhardt et al. (2002
) and
references cited therein. Mammalian NHEs are predicted to form 10-12
transmembrane (TM) helices with two distinct domains. The N-terminal half of
the protein contains all the TMs and is sufficient to transport H+
and cations. The current working model of membrane topology for NHEs
(Fig. 2) is largely based on
NHE1 (Wakabayashi et al.,
2000
). Future structure-function studies and approaches similar to
those described above would be necessary to determine whether this model is
applicable to all other NHEs as well.
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The cytoplasmic carboxy region modulates the overall function in response
to pHi and other regulatory factors. In the absence of the carboxy region,
high proton concentration (low pHi) is required for NHE1 activation. Thus,
intracellular protons bind to the sensor site, and the cytoplasmic region
integrates the `pH set point value' for NHE1 activation
(Orlowski and Grinstein,
1997). Several regulatory domains have been identified in
mammalian NHEs through which their activation is altered by several
interacting factors (Burckhardt et al.,
2002
).
Sequence and phylogenetic relationship of invertebrate NHEs
Molecular characterization of insect NHEs is now beginning. A cDNA with
high sequence similarity to NHE3 has been isolated from Aedes aegypti
(Table 1; Pullikuth et al.,
submitted; Hart et al., 2002).
Unlike vertebrate NHE3, Aedes NHE3 possesses a larger cytoplasmic
tail, accounting for nearly 56% of the protein. Aedes NHE3 lacks
potential N-glycosylation sites but does contain sites for potential
cAMP- and cGMP-dependent protein kinase phosphorylation. Protein kinase C and
casein kinase II substrate sites are also present in this isoform (Pullikuth
et al., submitted). Aedes NHE3 is 53% and 50% similar to
Drosophila NHE2 (Giannakou and
Dow, 2001
) and vertebrate NHE3
(Brant et al., 1995
),
respectively. We also identified NHE homologues in the draft genome sequence
of Anopheles gambiae. The genes for A. gambiae NHEs vary
significantly in size and exon usage (Table
1). Further searches revealed newer isoforms from
Drosophila in addition to the three reported by Giannakou and Dow
(2001
). High-throughput cDNA
sequencing of Drosophila cDNA libraries by Celera Genomics
(Rockville, MD, USA) indicated the presence of at least five distinct NHEs.
Consistent with this, our annotation of the Anopheles draft genome
also uncovered five distinct NHE-encoding genes. It is reasonable to assume
that insects might utilize five NHEs to effect cation/proton exchange.
However, it should be cautioned that NHE9 and NHE10 are at present only
assigned to the NHE family through their relatedness to NHE signature
sequence. These isoforms diverge considerably from the rest of the family and
are more closely related to bacterial NHEs, suggesting a possible horizontal
transfer event in evolution. Furthermore, they have a poorly conserved
amiloride-binding pocket, which is unusual among vertebrate members.
Alternatively, these isoforms could be expected to be insensitive to
pharmacological inhibition by agents conventionally used to distinguish
specific isoforms in vertebrates. A detailed knowledge on the expression
pattern and functional properties of these forms would be needed to
conclusively assign them to the invertebrate family of NHEs. The presence of
similar forms in vertebrates indicates the conservation of these molecules and
their function in evolution. Immunohistochemical studies of Aedes
NHE3 indicate that this isoform is predominantly expressed in the basolateral
membrane of midgut, Malpighian tubules and gastric ceca (Pullikuth et al.,
submitted). Distinct segments of the tubule exhibit apical, intracellular and
both basal and apical localization patterns for NHE3. This is consistent with
Petzel's suggestion that the basolateral Na+/H+ exchange
resembles the pharmacology of vertebrate NHE3
(Petzel, 2000
). However,
differential localization patterns might indicate its functional diversity at
specific segments of the tubule.
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In Drosophila, three NHEs (NHE1-3) were identified by Giannakou
and Dow (2001). Based on their
evolutionary relationship to well-characterized members of the vertebrate
family, we propose these Drosophila NHEs be assigned to NHE8, NHE3
and NHE6, respectively (Table
1). In addition, Drosophila cDNAs with high sequence
similarity to NHE9 and NHE10 have also been deposited in the database through
high-throughput cDNA sequencing efforts. It is important to note that both
Drosophila and Anopheles genomes do not contain members that
are closely related to mammalian NHE1, NHE2, NHE4, NHE5 and NHE7. The
physiological relevance of this finding is not clear, but it might indicate
functional redundancy by insect NHEs. Another possibility is that distinct
spliced isoforms may exist for insect NHEs, which might fulfill roles not
accomplished by the above forms. Drosophila, Caenorhabditis elegans
and Aedes NHEs have been shown to exist as spliced variants
(Giannakou and Dow, 2001
;
Hart et al., 2002
;
Nehrke and Melvin, 2002
). At
present, the specific roles of these variants are not known. However, it does
support the idea that functional diversity could be achieved by molecular
variants of a single gene in the NHE family in insects.
Anopheles NHE3 also possesses a large carboxy cytoplasmic region
containing several potential regulatory sites. Among the cloned NHEs, insect
NHE3s appear to be the largest with about 1200 amino acids. Potential splice
variants that lack the majority of the cytoplasmic tail, which contains
several potential phosphorylation sites, have been suggested to exist in
Aedes NHE3 (Hart et al.,
2002). However, the truncated isoform does retain a critical
phosphorylation site after the last predicted TM that appears to be analogous
to vertebrate Ser605, which is a target for cAMP-dependent inhibition of NHE3
(Kurashima et al., 1997
).
Conversely, in trout, ßNHE adjacent serines (Ser659 and Ser664) are
involved in cAMP-mediated activation of the exchanger
(Malapert et al., 1997
).
Anopheles NHE3 also contains dual serines at similar positions. It
would be interesting to determine the role of these residues in cAMP-dependent
processes in mosquito tubule function. As phosphorylation in the carboxy
region is one means of regulating the number of exchangers in the plasma
membrane, kinasing residues closer to the last TM would be expected to either
increase the number of exchangers on the plasma membrane (similar to trout
ßNHE; Malapert et al.,
1997
) or increase its endocytosis, thereby inactivating the
exchanger (similar to mammalian NHE3;
Kurashima et al., 1998
). Among
the insect NHEs, NHE8 appears to contain a sequence that is predicted to be
most sensitive to amiloride and its analogs (A. K. Pullikuth, K. Aimanova, W.
Kang'ethe and S. S. Gill, unpublished). These parameters render NHE8 a likely
candidate for the amiloride-sensitive exchanger in the apical membrane of
mosquito Malpighian tubules. To understand the function of this isoform, we
have recently cloned the NHE8 from Aedes and are currently
undertaking molecular studies to ratify this prediction (A. K. Pullikuth, K.
Aimanova, W. Kang'ethe and S. S. Gill, unpublished).
Sequence similarities and inhibitor sensitivity profiles of vertebrate NHEs
have been used to classify members of this family (reviewed in
Burckhardt et al., 2002). The
relationship of vertebrate and invertebrate NHEs is presented in
Fig. 3 based on deduced amino
acid sequences. Most vertebrate NHEs fall into a large clade where distinct
isoforms group in to separate branches of the phylogenetic tree. The organelle
forms, NHE6 and NHE7, form a separate branch that includes Anopheles
NHE6 and Drosophila NHE6 (previously assigned DmNHE3 by
Giannakou and Dow, 2001
).
Recently identified NHE8s are distantly related to the rest of the family,
whereas the novel forms NHE9 and NHE10 are more closely grouped with bacterial
NHEs. NHE9 and NHE10 are only tentatively assigned to the NHE family; their
conclusive inclusion as authentic members would require further functional
characterization. Moreover, the putative inhibitor-binding region is poorly
conserved in NHE9 and NHE10, raising the possibility that their function is
insensitive to currently used inhibitors.
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Cation-coupled chloride cotransporters (CCCs)
Structure and function of CCCs
Electroneutral transport of Na+ or K+ coupled with
Cl- can be separated into three distinct subtypes based on ion
specificity and pharmacology: Na+/K+/Cl-
cotransport, K+/Cl- cotransport and
Na+/Cl- cotransport. Characterization of these processes
in mammals facilitated the isolation of a group of membrane proteins
responsible for these activities, collectively named cation-coupled chloride
cotransporters (CCCs). Several excellent reviews on characterization of ion
cotransport in mammals and molecular and pharmacological studies of CCCs in
vertebrates have been published (Haas and
Forbush, 2000; Mount et al.,
1998
).
Sequence and structure analyses of CCCs revealed that they form a protein
family that is divergent from other ion transport proteins. Currently, eight
members of this family have been identified and functionally characterized in
vertebrates. Four isoforms are K+/Cl- transporters
(KCC1, KCC2, KCC3 and KCC4), two are
Na+/K+/2Cl- cotransporters (NKCC1 and NKCC2),
one is an Na+/Cl- cotransporter (NCC) and one is a
recently identified membrane protein, CIP1 (cotransporter interacting protein
1), which interacts with NKCC but does not transport ions by itself.
Theoretical hydrophobicity models and experimental data obtained by antibody
accessibility and protease-sensitivity analyses collectively predict that CCCs
possess 12 transmembrane segments, which are flanked by hydrophilic N- and
C-terminal cytoplasmic domains
(Gerelsaikhan and Turner,
2000; Moore-Hoon and Turner,
1998
).
CCCs are subdivided based on their ion specificities and different
sensitivities to loop diuretics. KCC isoforms are Na+ independent
and mediate K+ and Cl- cotransport. These cotransporters
can be stimulated by N-ethylmaleimide (NEM) and by the protein kinase
inhibitor staurosporine (Bize and Dunham,
1994). Although there are no inhibitors that reliably
differentiate between Na+-independent and Na+-dependent
K+/Cl- cotransporters, the alkaloid
(dihydroindenyl)oxyalkanoic acid is a more potent inhibitor of KCC than of
NKCC (Diecke and Beyer-Mears,
1997
). By contrast, the NCC protein provides
K+-independent Na+/Cl- cotransport and is
sensitive to thiazide diuretics (Costanzo,
1985
). The two NKCC proteins transport ions in a
1Na+:1K+: 2Cl- stoichiometry and are very
sensitive to bumetanide and other loop diuretics
(Haas and Forbush, 1998
).
Several reviews are available for detailed information on vertebrate CCCs (for
example, see Haas and Forbush,
2000
; Isenring and Forbush,
2001
; Mount et al.,
1998
).
Sequence and phylogenetic relationship of insect CCCs
In contrast to the substantial data on vertebrate CCCs, characterization of
insect members is limited. Only two insect CCC protein cDNAs have been cloned
so far, one in Manduca sexta
(Reagan, 1995) and another in
A. aegypti (Filippov et al.,
2003
), but no insect cotransporters have been functionally
characterized. Complete sequencing and annotation of two insect genomes,
Drosophila melanogaster and Anopheles gambiae, identified
five new CCC members in each of these species
(Adams et al., 2000
;
Holt et al., 2002
). To
evaluate relationships among the newly identified insect members of the CCC
family and characterized human cotransporters, we performed a phylogenetic
analysis of insect and human CCCs (Fig.
4). Protein sequences of eight known human CCCs were aligned with
12 insect CCCs identified in Drosophila, Manduca, Aedes and
Anopheles together with the sequence of human glycine transporter
type 2 (Evans et al., 1999
),
which was used as an outgroup member. This analysis showed that all insect CCC
proteins, with the exception of Drosophila CG5594, form their
separate branches, suggesting that they maintain higher levels of conservation
within Insecta compared with their potential vertebrate orthologues.
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The human CCC members form two major branches. One branch contains all human proteins involved in cotransport of Na+: NCC and two NKCCs. This branch also contains two clusters of insect proteins: the first one consists of Anopheles agCG57252 and its Drosophila homologue CG4357, while the second clusters the Manduca sexta NKCl cotransporter together with Drosophila CG31547 and two closely related proteins identified in Anopheles (agCG46536 and agCG46505). Clustering of these insect cotransporters in this branch might indicate that they are also sodium dependent in their ion cotransport across the cell membrane.
Another major branch of the phylogenetic tree has four human KCC isoforms
and the CIP1 protein. Drosophila CG5594 protein clusters confidently
with this branch and is the only insect protein used in the phylogenetic
analysis that shows considerable amino acid similarity to human members of the
CCC family (approximately 50% to KCC isoforms). The human CIP1 protein does
not transport ions itself but interacts with NKCC1
(Caron et al., 2000), forming
its own cluster within the KCC branch, which is more distant from human KCCs
than Drosophila CG5594. Two predicted insect proteins,
Drosophila CG10413 and Anopheles agCG54315, fall into this
branch. It would be interesting to determine whether these proteins are also
unable to transport ions themselves. This will mean that these proteins are
true orthologues of human CIP1 and that regulation by CCC heterodimerization
is an evolutionarily conserved way of changing kinetic and pharmacological
properties of ion cotransport.
Three insect proteins, Drosophila CG12773, Anopheles agCG52356 and Aedes AaeCG12773, form a branch that is equally distant from the two major branches of the tree. Since these CCCs are substantially divergent from mammalian members of the family, it is difficult to predict their cation requirements based on phylogenetic analysis. Uptake experiments using heterologous expression of the Aedes protein, which falls in this cluster, showed that its pharmacological properties are more related to the properties of human KCC isoforms than to other CCC members (V. Filippov and S. S. Gill, unpublished), suggesting that this cluster represents KCC proteins.
Transmembrane topology of insect CCC members predicted by several
theoretical methods showed results broadly similar to those found during
analysis of vertebrate cotransporters
(Gamba et al., 1994;
Gerelsaikhan and Turner,
2000
). As with mammalian CCCs, many prediction programs fail to
identify the last four membrane-spanning segments properly in insect CCCs and
instead predict two or three transmembrane domains in this region.
Experimental data obtained from mammalian cotransporters showed that these CCC
members have 12 transmembrane domains and their C- and N- ends are
intracellular (Haas and Forbush,
2000
). Based on comparative analysis of predicted topology of
mammalian and insect CCC members we can conclude that the latter also have 12
transmembrane domains. It is also interesting to note that predicted
intracellular regions are enriched in phosphorylation sites. In the case of
the Aedes cotransporter AaeCG12773, the longest
intracellular loop between transmembrane domains 10 and 11 contains eight out
of 12 predicted serine phosphorylation sites and four out of seven sites for
tyrosine phosphorylation (Filippov et al.,
2003
).
Phylogenetic and topological analyses of insect CCCs have evidently been very useful for initial assortment of these new members within the family. Being evolutionarily distant from the vertebrate CCC-encoding genes, the insect CCC members preserve the main structural characteristics necessary to execute similar functions, and this is reflected in the phylogenetic analysis. Comparison of insect CCCs from four different species (Drosophila, Anopheles, Aedes and Manduca) also revealed that they form separate clusters consisting of closely related members. Protein similarities within these clusters are considerably high (>50%; Table 2). The main tendency is that each mosquito protein has its own closely related homologue in Drosophila, and similarity in the primary sequences is also reflected in nearly identical predicted topology. There are two exceptions to this rule. First, as shown in Fig. 4, the Drosophila CG5594 does not have a similar Anopheles counterpart in the tree. However, the absence of a similar Anopheles gene is probably because the Anopheles genome is apparently not completely sequenced and annotated. Second, the Anopheles genome contains two similar annotated genes agCG46536 and agCG46505, whereas Drosophila has only one gene that falls in the cluster with them. Similarly, the presence of these two genes in the current annotation of the Anopheles genome could be due to heterogeneity in the Anopheles genome sequenced. Further analysis will be needed to confirm these initial results.
|
Studies of expression profiles and localization of CCCs in tissues proved
to be useful for determination of physiological roles of these cotransporters
in ion transport (Mount et al.,
1998; Mount and Gamba,
2001
). Sequence data now available for insect CCC members allow
the initiation of these studies in mosquitoes. Using RT-PCR, the expression
profile of the first cloned mosquito cotransporter gene, AaeCG12773,
has been obtained (Filippov et al.,
2003
). It was found that mRNA levels of this gene are most
abundant in the midgut and are also present in the hindgut, both in larvae and
adult females. No transcription of this gene was found in Malpighian tubules
(Filippov et al., 2003
).
Availability of antibodies against the AaeCG12773 protein allowed us not only
to confirm data obtained by RT-PCR but also to determine the subcellular
localization of the cotransporter. Immunostaining revealed that the AaeCG12773
protein is indeed predominantly localized in the upper part of the gut and its
expression is higher in adults compared with the larval stages
(Fig. 5). No specific
immunoreactivity above background was found in the Malpighian tubules.
Immunostaining also revealed that the protein is concentrated mainly in the
basolateral membranes.
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Table 2 summarizes the
available data so far on insect CCC members. They are arranged, based on
sequence similarity, in five groups. It is not obligatory that CCCs from
different species within the group are true orthologues, meaning that they
have identical physiological roles. However, it is likely that they do share
common ion preferences as well as pharmacological properties.
Drosophila is currently the only insect species in which
transcription profiles of all five CCC members are known. The mRNA levels of
these CCC-encoding genes differ considerably. One of these genes,
CG31547, showed such low levels of expression at all developmental
stages and in all tissues analyzed that its RT-PCR-specific band could not be
identified even after 70 cycles of amplification. It is interesting to note
that the Manduca cotransporter, which is in the same cluster as the
CG31547 gene, was found to be abundant in the Malpighian tubules
(Reagan, 1995). Other
Drosophila genes showed much higher expression levels. One important
finding is that high mRNA levels of four CCC-encoding genes were found during
embryonic development, including early embryos (0-4 h after deposition).
Expression of cotransporters at this stage definitely indicates the importance
of this type of ion transport for proliferation and/or differentiation. Three
CCC members, CG12773, CG4357 and CG5594, showed high levels
of expression in the larval midgut, suggesting that they are engaged in
regulation of ion transport during digestion. CG10413 showed high
expression levels in the larval brain; however, its expression dropped
substantially in adult heads. In contrast to CG10413, the
CG5594 gene, which has high similarity to human KCC isoforms, is not
transcribed in larval brains but its mRNA is abundant in adult heads. Analysis
of gene expression profiles of Drosophila CCCs also showed that,
compared with their mammalian homologues, none of them has high levels of
expression in Malpighian tubules.
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Considerations for models of transport in mosquito ion regulation |
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A major difference among vertebrate and invertebrate NHE function lies in
the substrate stoichiometry. Mammalian NHEs mediate the exchange of one proton
for each cation translocated and thus are electroneutral and membrane
potential insensitive (Aronson,
1989; Demaurex et al.,
1995
; Post and Dawson,
1994
). Interestingly, prokaryotes exhibit an electrogenic
transport of 1Na+/2H+
(Padan and Schuldiner, 1994
),
whereas crustacean transporters operate with a stoichiometry of
2Na+/1H+ (Ahearn et
al., 1994
). Due to the occurrence of two cation-binding sites,
crustacean exchangers are capable of transporting divalent cations
(Ca2+, Zn2+ and Cd2+) in exchange for protons
that may be required for sequestration and detoxification of heavy metals in
the hepatopancreas (Ahearn et al.,
2001
). Although electroneutral Na+/H+
exchange in invertebrates has been reported occasionally
(Deitmer and Schlue, 1987
;
Schlue and Thomas, 1985
),
electrogenic exchange of cations for protons is more widely distributed among
various groups of invertebrates (Grinstein
and Wieczorek, 1994
). Unlike vertebrate cells, where the
surrounding pH, ionic composition and buffering capacity do not vary
dramatically, bacteria and invertebrates experience wide ranges of pH and
ionic composition. In such cases, the chemical component itself would not be
sufficient for cellular homeostasis by driving electroneutral exchange, thus
the electrogenicity of cation/proton exchange could be more versatile in being
able to maintain the high luminal pH in the lepidopteran midgut (through
K+/2H+ exchange;
Lepier et al., 1994
) and in
the acidification of the gastric lumen in crustaceans (through
2Na+/1H+; Ahearn et
al., 1990
). The prevalence of electrogenic cation/proton exchange
in invertebrates has thus been viewed as an ancestral mechanism, whereas the
electroneutral mammalian exchange is considered an evolutionary adaptation
(Ahearn et al., 2001
;
Grinstein and Wieczorek,
1994
). In spite of these variations, Aedes aegypti
Malpighian tubules exhibit electroneutral exchange of
1K+/1H+ or 1Na+/1H+
(Weng et al., 2003
). The
tubule lumen is near neutral and thus would not require the electrogenic
apical cation/H+ exchange that has been implicated in maintaining
the alkaline lepidopteran or mosquito midgut lumen or the acidic lumen of
crustaceans. However, the electrogenic properties of this exchange under
stimulated conditions should be understood since cytoplasmic ionic
concentrations vary rapidly in blood-fed insects, which could conceivably
change the dynamics of apical transport.
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Summary and conclusions |
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Acknowledgments |
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References |
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