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First published online September 23, 2003

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Phylogeny and cloning of ion transporters in mosquitoes

Ashok K. Pullikuth, Valeri Filippov and Sarjeet S. Gill^*

Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA

View larger version (21K): [in a new window]   Fig. 1. Proposed model of ion transport in mosquito Malpighian tubules. Blood feeding initiates the release of diuretic peptides that trigger activation of ion channels in Malpighian tubules. Acute activation may result from a cAMP-mediated pathway controlling diuresis. The electrochemical gradient established by the vacuolar type-proton ATPase (V-ATPase) energizes secondary transport of intracellular ions and thus of fluid secretion through the apical membrane transporters. The secondary transporter(s) coupled to V-ATPase activity possesses pharmacological properties similar to sodium/proton exchangers (NHEs), whereas basolateral transport activated through cAMP is proposed to involve bumetanide-sensitive cation-coupled chloride cotransporters (CCCs). In the Malpighian tubules, the Na+/K+-ATPase is expressed on the basolateral membrane of proximal principal cells but not in distal principal cells.

View larger version (17K): [in a new window]   Fig. 2. Predicted membrane topology of a sodium/proton exchanger (NHE). NHEs are proposed to contain 12 transmembrane domains (N-domain) with varying regulatory elements in the carboxy termini. The N-domain is sufficient for transporting proton in exchange for Na+, whereas the carboxy cytoplasmic region determines several regulatory aspects of the exchanger. Vertebrate NHE1, modeled after Wakabayashi et al. (2000), is shown. CHP, PIP2 and CaM refer to schematic locations of domains implicated in calcineurin-homologous protein-binding, phosphatidyl inositide-binding and Ca2+/calmodulin-binding, respectively, in vertebrate NHE1. Aedes NHE3 possesses a nearly superimposable secondary structure (Pullikuth et al., submitted).

View larger version (32K): [in a new window]   Fig. 3. Phylogenetic relationship of insect sodium/proton exchangers (NHEs). Maximum parsimony analysis of representative NHEs from invertebrates and vertebrates indicates that most cloned vertebrate NHEs belong to the clade that contains mosquito NHE3. The organelle isoforms (NHE6, mitochondria and NHE7, TGN) form a distinct clade, whereas NHE8 isoforms are distantly related to characterized NHE members. The relationship of putative NHE9 and NHE10 to the rest of the family is unclear since it shares closer relationship to bacterial NHEs, which might suggest a possible horizontal transfer. Bootstrap values from 100 replications are given at nodes. Horizontal lines are proportional to the number of changes from the previous node. Accession numbers are given in parentheses. Invertebrate NHEs are shown in color. The original designation of Drosophila NHEs by Giannakou and Dow (2001) is given in parentheses next to their reassigned designations. The tree was rooted with E. coli NHE as outgroup.

View larger version (22K): [in a new window]   Fig. 4. Phylogenetic tree of insect and human cation-coupled chloride transporters (CCCs). The tree was constructed based on amino acid sequence alignment by clustal method using the alignX program in the Vector NTI package (InforMax, Bethesda, MD, USA). Phylogenetic analysis was performed using the PAUP v4.0b10 program (Rogers and Swofford, 1998). The results obtained were transformed by the TREEVIEW program (Page, 1996) for final display. The human glycine transporter type 2 protein sequence marked as SLC6A5 (Evans et al., 1999) was used as an outgroup member in this analysis. NKCl Mse represents the cotransporter isolated in Manduca sexta. All human CCC members have suffixes of hum, while Anopheles gambiae proteins are prefixed by agCG, Drosophila melanogaster by CG and Aedes aegypti by AaeCG.

View larger version (110K): [in a new window]   Fig. 5. Immunolocalization of the AaeCG12773 cotransporter in the mosquito digestive system. (A) The gut and Malpighian tubules of a 4th instar larva of Aedes aegypti. Specific immunostaining is detected in gastric ceca (CA), anterior (AMG) and posterior (PMG) midgut and pylorus (P). Immunofluorescence in the Malpighian tubules (MT) and rectum (R) is low and does not exceed background levels of pre-immune serum. (B,C) Immunohistochemistry of sections of a 4th instar A. aegypti larva. Subcellular localization of the AaeCG12773 cotransporter by specific antibodies clearly shows that it is predominantly present in the basolateral membrane of the midgut. No specific immunoreactivity was observed in sectioned Malpighian tubules and rectum.