Inhibition of Sodium–Hydrogen Antiport by Antibodies to NHA1 in Brush Border Membrane Vesicles from Whole Aedes aegypti Larvae
- 50 Downloads
The present research report describes Na+/H+ antiport by brush border membrane vesicles isolated from whole larvae of Aedes aegypti (AeBBMVw). Our hypothesis is that acid quenching of acridine orange by AeBBMVw is predominantly mediated by Na+/H+ antiport via the NHA1 component of the AeBBMVw in the absence of amino acids and ATP. AeNHA1 is a Na+/H+ antiporter that has been postulated to exchange Na+ and H+ across the apical plasma membrane in posterior midgut of A. aegypti larvae. Its principal function is to recycle the H+ and Na+ that are transported during amino acid uptake, e.g., phenylalanine. This uptake is mediated, in part, by a voltage-driven, Na+-coupled, nutrient amino acid transporter (AeNAT8). The voltage is generated by an H+ V-ATPase. All three components, V-ATPase, antiporter, and nutrient amino acid transporter (VAN), are present in brush border membrane vesicles isolated from whole larvae of A. aegypti. By omitting ATP and amino acids, Na+/H+ antiport was measured by fluorescence quenching of acridine orange (AO) caused by acidification of either the internal vesicle medium (Na+in > Na+out) or the external fluid-membrane interface (Na+in < Na+out). Vesicles with 100 micromolar Na+ inside and 10 micromolar Na+ outside or with 0.01 micromolar Na+ inside and 100 micromolar Na+ outside quenched fluorescence of AO by as much as 30%. Acidification did not occur in the absence of AeBBMVw. Preincubation of AeBBMVw with antibodies to NHA1 inhibit Na+/H+ antiport dependent fluorescence quenching, indicating that AeNHA1 has a significant role in Na+/H+ exchange.
KeywordsAeNHA1 AeNAT8 H+ V-ATPase Electrophoretic Acridine orange Acidification Fluorescence-quench
Aedes aegypti brush border membrane vesicles from whole larvae
Other membrane proteins
N-Aminopeptidase/Bacillus thuringiensis israelensis (Bti) receptor
Na+/H + antiporter
Nutrient amino acid transporter
Na+/H + exchanger
V-ATPase, antiporter, nutrient amino acid transporter
We thank Linda Greene for assistance with the fluorescence plate reader. This research was supported in part by facilities and funds from the Whitney Laboratory, Peter A.V. Anderson, director emeritus, and funds from Barbara H. Mayer.
Compliance with Ethical Standards
Conflict of interest
Kenneth M. Sterling has received no research grant from any company or individual and declares that he has no conflict of interest. William R. Harvey received funds from Barbara H. Mayer and he declares that he has no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.
Research Involving Animal Studies
4th instar Aedes aegypti larvae were used for this study.
- Ahearn GA, Grover ML, Dunn RE (1985) Glucose transport by lobster hepatopancreatic brush-border membrane vesicles. Am J Physiol 248:R133–R141Google Scholar
- Becnel JJ (1997) Complementary techniques: preparations of entomopathogens and diseased specimens for more detailed study using microscopy. In: Lacey LA (ed) Manual of techniques in insect pathology. Academic Press, New YorkGoogle Scholar
- Behnke RD, Busquets-Turner L, Ahearn GA (1998) Epithelial glucose transport by lobster antennal gland. J Exp Biol 201:3385–3393Google Scholar
- Clements AN (1992) The biology of mosquitoes. Chapman and Hall Press, LondonGoogle Scholar
- Cornish-Bowden A (1999) Fundamentals of enzyme kinetics. Princeton University Press, PrincetonGoogle Scholar
- Dowd JE, Riggs DS (1965) A comparison of estimates of michaelis-menten kinetic constants from various linear transformations. J Biol Chem 240:863–869Google Scholar
- Eadie GS (1942) The inhibition of cholinesterase by physostigmine and prostigmine. J Biol Chem 146:85–93Google Scholar
- Hearn PR, Russell RGG, Farmer J (1981) The formation and orientation of brush border vesicles from rat duodenal mucosa. J Cell Sci 47:227–236Google Scholar
- Kell DB (1979) On the functional proton current pathway of electron transport phoshphorylation: an electrodic view. Biochem Biophys Acta 549:55–99Google Scholar
- Leonardi MG, Caccia S, Giordana B (2006) Brush border membrane vesicles from dipteran midgut: a tool for studies on nutrient absorption. ISJ 3:137–145Google Scholar
- Mitchell D (2014) Help topics for exploratory enzyme kinetics. In: SYSTAT, pp. 1–17Google Scholar
- Michaelis L, Menten ML (1913) Die Kinetik der Invertinwirkung. Biochemische Zeitschrift 49:333–369Google Scholar
- Suzuki T, Kishi Y, Totani M, Kagamiyama H, Murachi T (1987) Monoclonal and polyclonal antibodies against porcine mitochondrial aspartate aminotransferase: their inhibition modes and application to enzyme immunoassay. Biotechnol Appl Biochem 9:170–180Google Scholar
- Taglicht D, Padan E, Schuldiner S (1993) Proton-sodium stoichiometry of NhaA, an electrogenic antiporter from Escherichia coli. J Biol Chem 268(8):5382–5387Google Scholar
- Warnock DG, Reenstra WW, Yee VJ (1982) Na+/H + antiporter of brush border vesicles: studies with acridine orange uptake. Am J Physiol 241:F733–F739Google Scholar
- Wieczorek H, Putzenlechner M, Zeiske W, Klein U (1991) A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane. J Biol Chem 266:15340–15347Google Scholar
- Wolfersberger M, Luethy P, Maurer A, Parenti P, Sacchi FV, Giordana B, Hanozet GM (1987) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp Biochem Physiol 86A:301–308CrossRefGoogle Scholar
- Zhuang Z, Linser PJ, Harvey WR (1999) Antibody to H(+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti). J Exp Biol 202:2449–2460Google Scholar