Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan,Anopheles gambiae

Abstract

SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes(Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned,nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities

“…Along with a sequenced genome and uniquely potent genetic tools, functional transport readouts can be provided by the simple renal system (11,12). As in humans and the malaria vector mosquito Anopheles gambiae (13,14), the fruit fly Drosophila has two NHA genes, Nha1 and Nha2 (5). The NHA family is relatively divergent, however, and asserting orthology between human and insect NHAs is difficult (5); in terms of sequence identity, insect NHA1 is slightly more similar to human NHA2, and vice versa.…”

A comprehensive transcriptomic view of renal function in the malaria vector, Anopheles gambiae

“…The data further show that the larval and adult tubules use the same subset of V-ATPase genes to encode the plasma membrane V-ATPase (Table 2). Similarly, the Na + , K + ATPase (Patrick and Gill, 2002), the aquaporin DRIP (Liu et al, 2011) and the apical exchanger (Rheault et al, 2007;Xiang et al, 2011;Xiang et al, 2012b) have all been described in mosquito tubule; these array data provide a more comprehensive survey of which genes in a particular family show highly enriched expression in the tubule.…”

Protein kinase A dependent and independent activation of the V-ATPase in Malpighian tubules ofAedes aegypti.

“…Although we cannot fully exclude that activation of the V-ATPase in Malpighian tubules of A. aegypti depends on direct phosphorylation of the enzyme, another activation mechanism appears conceivable. A potential target of PKA could be the apical Na ( Rheault et al, 2007;Day et al, 2008), which upon phosphorylation might increase its activity, thereby contributing to an acidification of the cytosol in principal cells. This pH drop could then induce the assembly of V 1 and V O complexes and hence increase transepithelial ion secretion.…”