Meeting Abstract

P2.83  Friday, Jan. 4  Identification and localization of NHE8 in Malpighian tubules of the yellow fever mosquito Aedes aegypti WEIHRAUCH, D.*; PIERMARINI, P.M.; BEYENBACH, K.W.; University of Manitoba, Winnipeg; Cornell University, Ithaca; Cornell University, Ithaca; U of Osnabrueck, Osnabruck weihrauc@cc.umanitoba.ca

Current models of transepithelial electrolyte secretion in Malpighian tubules of insects include a hypothetical cation/H+ exchange transporter in the apical membrane of principal cells. An NHE isoform related to the SLC9 superfamily of transporters is thought to mediate the extrusion of Na+ and K+ from cell to tubule lumen in exchange for H+. In the present study we have cloned the cDNA encoding an NHE from Malpighian tubules of adult yellow fever mosquitoes Aedes aegypti. The cDNA sequence was obtained using the methods of RT-PCR and RACE with primers gleaned from a predicted Anopheles NHE. The predicted protein has 668 amino acids and a molecular weight of 73.5 kDa. The amino-acid sequence is identical to that of AeNHE8 determined by Kang�ethe et al. (Am J Physiol Renal 292: 1501-1512). Among insect NHEs, AeNHE8 shares the highest amino-acid identity (83%) with an Anopheles NHE (EAA03626), followed by Drosophila and Apis NHE1 (65%). Among mammalian NHEs, AeNHE8 is most identical (> 50%) to isoform 8. Similar to other SLC9-like proteins, membrane-topology predictions suggest that AeNHE8 is composed of 12 to 13 transmembrane segments with approximately 10 amino acids forming a short NH2-terminal domain in the cytoplasm, and 165 residues forming a long cytoplasmic, COOH terminus. An antibody raised against AeNHE8 detects immunoreactivity in the sub-apical vesicular region of principal cells, and not in V-type ATPase-rich brush border. These localization results indicate that if AeNHE8 is responsible for apical cation/H+ exchange in principal cells, then the transporter must be trafficked to the apical membrane.