H(+) V-ATPase-energized transporters in brush border membrane vesicles from whole larvae of Aedes aegypti.
Autor(es): Harvey William R; Okech Bernard A; Linser Paul J; Becnel James J; Ahearn Gregory A; Sterling Kenneth M
Resumo: Brush border membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs) contain an H(+) V-ATPase (V), a Na(+)/H(+) antiporter, NHA1 (A) and a Na(+)-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from Anopheles gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but approximately 10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4h. Na(+)-coupled (3H)phenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na(+) currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.
Palavras-Chave: BBMV; H+ V-ATPase; NHA1; NAT8; VAN
Imprenta: Journal of Insect Physiology, v. 56, n. 10, p. 1377-1389, 2010
Identificador do objeto digital: 10.1016/j.jinsphys.2010.04.017
Descritores: Aedes aegypti - Cell ; Aedes aegypti - Genome ; Aedes aegypti - Molecular Structure ; Aedes aegypti - Pathogenesis ; Aedes aegypti - Proteins ; Aedes aegypti - Antibodies
Data de publicação: 2010