Location: Mosquito and Fly ResearchTitle: High affinity 3H-Phe uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBMVw)) Author
Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2012
Publication Date: 4/1/2012
Citation: Sterling, K.M., Okech, B.A., Xiang, M.A., Linser, P.J., Price, D.A., Vanekeris, L., Becnel, J.J., Harvey, W.R. 2012. High affinity 3H-phenylalanine uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBBMVw). Journal of Insect Physiology. 58:580-589. Interpretive Summary: There is a critical need for the development of new larvicides for control of mosquitoes that vector diseases to man and animals but are safe for non-target organisms and have minimal environmental impacts. The ability to study and discover new target sites in the larval midgut is difficult due to their small size. In this collaborative research project between the Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology in Gainesville, FL and University of Florida researchers, a method has been developed to isolate large quantities of midgut components (brush border membranes) from larvae of the yellow fever mosquito Aedes aegypti. This technology can be utilized to conduct detailed studies on midgut transporters and receptors that can be targeted for the development of novel larvicides.
Technical Abstract: Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to study Na+-coupled, 3H-labeled, phenylalanine (Phe) uptake. The affinity for all components of the uptake was very high with half maximal values in the sub-micromolar range. By contrast a KPhe 0:5 of 0.2 mM and a KNa 0:5 of 26 mM were calculated from Phe-induced electrical currents in Xenopus oocytes that were heterologously expressing the Anopheles gambiae symporter (co-transporter), AgNAT8, in a buffer with 98 mM Na+. What accounts for the >1000-fold discrepancy in affinity for substrates between the BBMV and oocyte experiments? Is it because Ae. aegypti were used to isolate BBMVw whereas An. gambiae were used to transfect oocytes? More likely, it is because BBMVw were exposed to [Na+] in the micromolar range with the transporter(s) being surrounded by native lipids. By contrast, the oocyte measurements were made at [Na+] 100,000 times higher with AgNAT8 surrounded by foreign frog lipids. The results show that AaBBMVw are osmotically sealed; the time-course has a Na+-induced overshoot, the pH optimum is _7 and the K0.5 values for Phe and Na+ are very low. The transport is virtually unchanged when Na+ is replaced by K+ or Li+ but decreased by Rb+. This approach to resolving discrepancies between electrical data on solute transporters such as AgNAT8 that are over-expressed in oocytes and flux data on corresponding transporters that are highly expressed in native membrane vesicles, may serve as a model for similar studies that add membrane biochemistry to molecular biology in efforts to identify targets for the development of new methods to control disease-vector mosquitoes.