H V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes aegypti |
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Authors: | William R Harvey Bernard A Okech Paul J Linser Gregory A Ahearn |
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Institution: | a Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA b Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32610, USA c Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610, USA d Department of Environmental and Global Health, University of Florida, Gainesville, FL 32610, USA e U.S. Department of Agriculture, Agricultural Research Service Center for Medical, Agricultural and Veterinary Entomology, Gainesville, FL 32608, USA f University of North Florida, Jacksonville, FL 32224, USA |
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Abstract: | 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 ∼10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4 h. Na+-coupled 3Hphenylalanine 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. |
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Keywords: | BBMV H+ V-ATPase NHA1 NAT8 VAN |
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