Hematophagous insects such as female mosquitoes have a remarkably fast and efficient excretion system that allows rapid removal of the enormous sodium load acquired during bloodfeeding to restore homeostasis. Previous electrophysiological studies on Malpighian tubules - the major renal organ in insects - have postulated a role for an amiloride sensitive Na+ /H+ exchanger extruding cations into the lumen driven secondarily by the proton gradient created by a H+-ATPase in the apical membrane. We have identified the implicated exchanger in Aedes aegypti and designated it AeNHE8. Immunolocalization studies demonstrated its expression in the apical membranes of Malpighian tubules, gastric caecae and rectum. Upon heterologous expression in yeast cells lacking Na+ extrusion proteins, AeNHE8 rescues their salt-sensitive phenotype and also allows the cells to tolerate hygromycin-B. Additionally, heterologous expression in NHE-deficient mammalian fibroblast cells results in amiloride sensitive 22Na + uptake. To determine the exchanger's kinetic properties, we reconstituted membranes from yeast cells expressing the protein into proteoliposomes and assayed for cation-dependent H+ exchange by fluorimetric methods. Our results indicated that it mediates saturable exchange of Na+ and K+ for H+. We further conducted a mutational analysis of AeNHE8 expressed in yeast cells and found that the first transmembrane domain is not essential for Na+ transport and hygromycin-B tolerance. In contrast, the C-terminal cytoplasmic domain is critical for Na+ transport, but dispensable for Hygromycin-B tolerance, indicating that the domains mediating these two signature activities could be distinct or separable. We also performed a yeast-2-hybrid screen and found that the C-terminal fragment interacted with a putative E3 ubiquitin ligase; coincidentally, NHE8 abundance in the apical membrane of principal cells varied with bloodfeeding. And most interestingly perhaps, its apparent mass increases by approximately 2--4kDa following bloodfeeding. These observations point to a possible post-translational regulatory mechanism for the exchanger that resembles the ubiquitin-mediated trafficking of ENaC in mammals. Finally, we carried out RNAi-mediated NHE8 knockdown using the Gal4/UAS binary system in Drosophila and found that the larvae's ability to tolerate high salt was severely compromised.;In sum, we have dissected AeNHE8 in molecular detail and shown it to be critical for sodium excretion in dipteran insects.
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