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

Abstract

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-sensitive22Na+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.

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

“…This discrepancy suggests that vertebrate NHE8s may function differently from insect NHE1s. Expression of AgNHE1 was not detected in the midgut epithelium of Aedes, however, and therefore it is not a likely candidate for the putative electrophoretic K + /2H + antiporter that is discussed below (Kang'ethe et al, 2007).…”

“…However, since the mid-1980s, H + V-ATPases have been recognized as a second major class of animal plasma membrane energizers, which set up voltage gradients (cell negative) that drive secondary electrophoretic transporters (for reviews, see Wieczorek et al, 1999;Nelson and Harvey, 1999). In the postgenomic era the two ATPases and several electroneutral NHEs have been cloned and characterized from numerous organisms (Hart et al, 2002;Kang'ethe et al, 2007;Orlowski and Grinstein, 1997;Orlowski and Grinstein, 2004;Pullikuth et al, 2006) but the 'other shoe has never dropped' -no NHA has previously been cloned and localized in a metazoan organism, although they are present in every phylum for which a genome is available.…”

“…NHE8 is widely expressed in vertebrate cells, where it is localized in plasma and endosomal membranes (Goyal et al, 2005;Orlowski and Grinstein, 2004). By contrast Aedes aegypti AeNHE1 (formerly AeNHE8), which is a homolog of vertebrate NHE8, is expressed solely in the apical membranes of the gastric caeca, Malpighian tubules and rectum (Kang'ethe et al, 2007). This discrepancy suggests that vertebrate NHE8s may function differently from insect NHE1s.…”

“…The pH approaches 11 in the anterior midgut, drops to 9.5 in middle midgut and falls rapidly to values from 6.2 to 7 in posterior midgut (Clements, 1992). The cloning and localization of AeNHE1 (Kang'ethe et al, 2007) and AeNHE2 (Pullikuth et al, 2006) from adult Ae. aegypti and that of AgNHA1 from larval An.…”

“…Evidence that electroneutral Na + /H + exchangers (NHE) are present in epithelia and neural tissues of invertebrates is available from molecular cloning studies (Gaillard and Rodeau, 1987;Kang'ethe et al, 2007;Pullikuth et al, 2006;Schlue and Thomas, 1985;Strauss and Graszynski, 1992). By contrast, evidence for electrophoretic Na + /H + antiporters (NHA) is derived only from functional studies on isolated membrane vesicles.…”