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NHE8 mediates amiloride-sensitive Na⁺/H⁺ exchange across mosquito Malpighian tubules and catalyzes Na⁺ and K⁺ transport in reconstituted proteoliposomes

¹Graduate Program in Environmental Toxicology, ²Department of Cell Biology and Neuroscience, University of California, Riverside, California

Submitted 7 December 2005 ; accepted in final form 26 January 2007

Following a blood meal, the mosquito Aedes aegypti will have acquired an enormous sodium load that must be rapidly excreted to restore ion homeostasis. It is a process that demands robust sodium and fluid transport capabilities. Even though the identities of the components involved in this ion transport across the mosquito Malpighian tubule epithelia have not been completely determined, electrophysiological studies suggest the contribution of a Na⁺/H⁺ exchanger extruding cations into the lumen driven secondarily by the proton gradient created by the V-type H⁺-ATPase in the tubules' apical membrane. We have identified the putative exchanger and designated it AeNHE8. Immunolocalization studies demonstrated that AeNHE8 is expressed in the apical membranes of Malpighian tubules, gastric caecae, and rectum. When heterologously expressed in salt-sensitive yeast cells lacking Na⁺ extrusion and Na⁺/H⁺ exchange proteins, AeNHE8 rescues the salt-sensitive phenotype and restores the cells' ability to grow in high NaCl media. Furthermore, heterologous expression of AeNHE8 in NHE-deficient fibroblast cells results in an amiloride-sensitive ²²Na⁺ uptake. To determine the exchanger's kinetic properties, we reconstituted membranes from yeast cells expressing the protein into lipid proteoliposomes and assayed for cation-dependent H⁺ exchange by fluorimetric methods. Our results indicate that AeNHE8 mediates saturable exchange of Na⁺ and K⁺ for H⁺. We propose that AeNHE8 may be coupled to the inward H⁺ gradient across the Malpighian tubules and plays a role in the extrusion of excess sodium and potassium while maintaining steady intracellular pH in the principal cells.

ion homeostasis; Aedes aegypti; apical membrane

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