The Journal of Membrane Biology

, Volume 252, Issue 1, pp 1–16 | Cite as

Inhibition of Sodium–Hydrogen Antiport by Antibodies to NHA1 in Brush Border Membrane Vesicles from Whole Aedes aegypti Larvae

  • Kenneth M. SterlingEmail author
  • William R. Harvey


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.


AeNHA1 AeNAT8 H+ V-ATPase Electrophoretic Acridine orange Acidification Fluorescence-quench 



Aedes aegypti brush border membrane vesicles from whole larvae


Other membrane proteins


Acridine orange


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid




4-Morpholinepropanesulfonic acid




Gastric caeca


Anterior midgut


Posterior midgut


Malpighian tubules


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.

Ethical Approval

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.


  1. Abdul-Rauf M, Ellar DJ (1999) Isolation and characterization of brush border membrane vesicles from whole Aedes aegypti larvae. J Invertebr Pathol 73:45–51CrossRefGoogle Scholar
  2. Ahearn GA, Grover ML, Dunn RE (1985) Glucose transport by lobster hepatopancreatic brush-border membrane vesicles. Am J Physiol 248:R133–R141Google Scholar
  3. Aslamkhan A, Han YH, Walden R, Sweet DH, Pritchard JB (2003) Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells. Am J Physiol Renal Physiol 285:F775–F783CrossRefGoogle Scholar
  4. Azuma M, Harvey WR, Wieczorek H (1995) Stoichiometry of K+/H+ antiport helps to explain extracellular pH 11 in a model epithelium. FEBS Lett 361:153–156CrossRefGoogle Scholar
  5. 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
  6. Becnel JJ, White SE, Moser BA, Rotstein MJ, Undeen AH, Cockburn A (2001) Epizootiology and transmission of a newly discovered baculovirus from the mosquitoes Culex nigripalpus and Culex quinquefasciatus. J Gen Virol 82:275–282CrossRefGoogle Scholar
  7. Behnke RD, Busquets-Turner L, Ahearn GA (1998) Epithelial glucose transport by lobster antennal gland. J Exp Biol 201:3385–3393Google Scholar
  8. Beyenbach KW, Wieczorek H (2006) The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J Exp Biol 209:577–589CrossRefGoogle Scholar
  9. Booth AG, Kenny AJ (1974) A rapid method for the preparation of microvilli from rabbit kidney. Biochem J 142:575–581CrossRefGoogle Scholar
  10. Boudko DY, Moroz LL, Harvey WR, Linser PJ (2001) Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut. Proc Natl Acad Sci USA 98:15354–15359CrossRefGoogle Scholar
  11. Brett CL, Donowitz M, Rao R (2005) Evolutionary origins of eukaryotic sodium/proton exchangers. Am J Physiol Cell Physiol 288:C223–C239CrossRefGoogle Scholar
  12. Casadio R (1991) Measurements of transmembrane pH differences of low extents in bacterial chromatophores. Eur Biophys J 19:189–201CrossRefGoogle Scholar
  13. Chen JW, Aimanova KG, Pan SQ, Gill SS (2009) Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin. Insect Biochem Mol Biol 39:688–696CrossRefGoogle Scholar
  14. Chintapalli VR, Kato A, Henderson L, Hiratab T, Woods DJ, Overend G, Davies SA, Romero MF, Dow JA (2015) Transport proteins NHA1 and NHA2 are essential for survival but have distinct transport modalities. Proc Natl Acad Sci USA 112:11720–11725CrossRefGoogle Scholar
  15. Cioffi M (1984) Comparative ultrastructure of arthropod transporting epithelia. Am Zool 24:139–156CrossRefGoogle Scholar
  16. Clements AN (1992) The biology of mosquitoes. Chapman and Hall Press, LondonGoogle Scholar
  17. Cornish-Bowden A (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J 137:143–144CrossRefGoogle Scholar
  18. Cornish-Bowden A (1999) Fundamentals of enzyme kinetics. Princeton University Press, PrincetonGoogle Scholar
  19. Cornish-Bowden A, Eisenthal R (1974) Statistical considerations in the estimation of enzyme kinetic parameters by the direct linear plot and other methods. Biochem J 139:721–730CrossRefGoogle Scholar
  20. Cortés A, Cascante M, Cárdenas ML, Cornish-Bowden A (2001) Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data. Biochem J 357:263–268CrossRefGoogle Scholar
  21. Day JP, Wan S, Allan AK, Kean L, Davies SA, Gray JV, Dow JA (2008) Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa. J Cell Sci 121:2612–2619CrossRefGoogle Scholar
  22. Dixon M (1953) The determination of enzyme inhibitor constants. Biochem J 55:170–171CrossRefGoogle Scholar
  23. Donini A, O’Donnell MJ (2005) Analysis of Na+, Cl, K+, H+ and NH4 + concentration gradients adjacent to the surface od anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. J Exp Biol 208:603–610CrossRefGoogle Scholar
  24. 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
  25. D’Silva NM, Patrick ML, O’Donnell MJ (2017) Effects of rearing salinity on expression and function of ion transport across the gastric caecum of Aedes aegypti larvae. J Exp Biol 220:3172–3180CrossRefGoogle Scholar
  26. Eadie GS (1942) The inhibition of cholinesterase by physostigmine and prostigmine. J Biol Chem 146:85–93Google Scholar
  27. Eisenthal R, Cornish-Bowden A (1974) The direct linear plot: a new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139:715–720CrossRefGoogle Scholar
  28. Farady CJ, Sun J, Darragh MR, Miller SM, Craik CS (2007) The Mechanism of Inhibition of Antibody-based Inhibitors of membrane-type Serine Protease 1 (MT-SP1). J Mol Biol 369:1041–1051CrossRefGoogle Scholar
  29. Gonzales KK, Hansen IA (2016) Artificial diets for mosquitoes. Int J Environ Res Public Health 12:1267-CrossRefGoogle Scholar
  30. Gruber G, Radermacher M, Ruiz T, Godovac-Zimmermann J, Canas B, Kleine-Kohlbrecher D, Huss M, Harvey WR, Wieczorek H (2000) Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut. Biochemistry 39:8609–8616CrossRefGoogle Scholar
  31. Grzesiek S, Otto H, Dencher NA (1989) ∆pH-induced fluorescence quenching of 9-aminoacridine in lipid vesicles is due to excimer formation at the membrane. Biophys J 55:1101–1109CrossRefGoogle Scholar
  32. Haase W, Schafer A, Murer H, Kinne R (1978) Studies on the orientation of Brush-Border membrane vesicles. Biochem J 172:57–62CrossRefGoogle Scholar
  33. Hanes CS (1932) Studies on plant amylases: the effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem J 26(5):1406–1421CrossRefGoogle Scholar
  34. Harvey WR (2009) Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters. J Exp Biol 212:1620–1629CrossRefGoogle Scholar
  35. Harvey WR, Boudko DY, Rheault MR, Okech BA (2009) NHEVNAT: an H+ V-ATPase electrically coupled to a Na+: nutrient amino acid transporter (NAT) forms an Na+/H+ exchanger (NHE). J Exp Biol 212:347–357CrossRefGoogle Scholar
  36. Harvey WR, Cioffi M, Wolfersberger MG (1981) Portasomes as Coupling factors in active ion transport and oxidative phosphorylation. Am Zool 21:775–791CrossRefGoogle Scholar
  37. Harvey WR, Nedergaard S (1964) Sodium-independent active transport of potassium in the isolated midgut of Cecropia silkworm. Proc Natl Acad Sci USA 51:757–765CrossRefGoogle Scholar
  38. Harvey WR, Okech BA (2010) H+, Na+, K+ and Amino Acid Transport in Caterpillar and Larval Mosquito Alimentary Canal. In: Gerencser GA (ed) Epithelial transport physiology. Springer, New York, pp 113–148CrossRefGoogle Scholar
  39. Harvey WR, Okech BA, Linser PJ, Becnel JJ, Ahearn GA, Sterling KM (2010) H(+) V-ATPase-energized transporters in brush border membrane vesicles from whole larvae of Aedes aegypti. J Insect Physiol 56:1377–1389CrossRefGoogle Scholar
  40. 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
  41. Hofstee BHJ (1959) Non-inverted versus inverted plots in enzyme kinetics. Nature 184(4695):1296–1298CrossRefGoogle Scholar
  42. Hutzler JM, Tracy TS (2002) Atypical kinetic profiles in drug metabolism reactions. Drug Metab Dispos 30:355–362CrossRefGoogle Scholar
  43. Kell DB (1979) On the functional proton current pathway of electron transport phoshphorylation: an electrodic view. Biochem Biophys Acta 549:55–99Google Scholar
  44. 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
  45. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658CrossRefGoogle Scholar
  46. MacIntosh SC, Lidster BD, Kirkham CL (1994) Isolation of brush border membrane vesicles from whole diamondback moth (Lepidoptera: Plutellidae) larvae. J Invertebr Pathol 63:97–98CrossRefGoogle Scholar
  47. Meleshkevitch EA, Assis-Nascimento P, Popova LB, Miller MM, Kohn AB, Phung EN, Mandal A, Harvey WR, Boudko DY (2006) Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family. J Exp Biol 209:3183–3198CrossRefGoogle Scholar
  48. Mitchell D (2014) Help topics for exploratory enzyme kinetics. In: SYSTAT, pp. 1–17Google Scholar
  49. Mitchell P (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144–148CrossRefGoogle Scholar
  50. Moriyama Y, Takano T, Ohkuma S (1982) Acridine orange as a fluorescent probe for lysosomal proton pump. J Biochem 92:1333–1336CrossRefGoogle Scholar
  51. Murer H, Ahearn G, Amstutz M, Biber J, Brown C, Gmaj P, Hagenbuch B, Malmstrom K, Mohrmann I, Mohrmann M et al (1985) Cotransport systems for inorganic sulfate and phosphate in small intestine and renal proximal tubule. Ann NY Acad Sci 456:139–152CrossRefGoogle Scholar
  52. Murer H, Hopfer U, Kinne R (1976) Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney. Biochem J 154:597–604CrossRefGoogle Scholar
  53. Michaelis L, Menten ML (1913) Die Kinetik der Invertinwirkung. Biochemische Zeitschrift 49:333–369Google Scholar
  54. Michaelis L, Menten ML (2013) (Submitted 4 February 1913, Translated by T.R.C. Boyde). The kinetics of invertin action. FEBS Lett 587:2712–2720CrossRefGoogle Scholar
  55. Munishkina LA, Fink AL (2007) Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins. Biochimica et Biophysica Acta 1768:1862–1885CrossRefGoogle Scholar
  56. Nelson N, Harvey WR (1999) Vacuolar and plasma membrane proton-adenosinetriphosphatases. Physiol Rev 79:361–385CrossRefGoogle Scholar
  57. Okech BA, Boudko DY, Linser PJ, Harvey WR (2008) Cationic pathway of pH regulation in larvae of Anopheles gambiae. J Exp Biol 211:957–968CrossRefGoogle Scholar
  58. Orlowski J, Grinstein S (2004) Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Arch 447:549–565CrossRefGoogle Scholar
  59. Padan E, Bibi E, Ito M, Krulwich TA (2005) Alkaline pH homeostasis in bacteria: new insights. Biochim Biophys Acta 1717:67–88CrossRefGoogle Scholar
  60. Patrick ML, Aimanova K, Sanders HR, Gill SS (2006) P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti. J Exp Biol 209:4638–4651CrossRefGoogle Scholar
  61. Piermarini PM, Weihrauch D, Meyer H, Huss M, Beyenbach KW (2009) NHE8 is an intracellular cation/H+ exchanger in renal tubules of the yellow-fever mosquito Aedes aegypti. Am J Physiol Renal Physiol 296:F730–F750CrossRefGoogle Scholar
  62. Rheault MR, Okech BA, Keen SB, Miller MM, Meleshkevitch EA, Linser PJ, Boudko DY, Harvey WR (2007) Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan, Anopheles gambiae. J Exp Biol 210:3848–3861CrossRefGoogle Scholar
  63. Skou JC (1990) The energy coupled exchange of Na+ for K+ across the cell membrane—The Na+, K+-pump. FEBS Lett 268:314–324CrossRefGoogle Scholar
  64. Sterling KM Jr, Cheeseman CI, Ahearn GA (2009) Identification of a novel sodium-dependent fructose transport activity in the hepatopancreas of the Atlantic lobster Homarus americanus. J Exp Biol 212:1912–1920CrossRefGoogle Scholar
  65. Sterling KM, Okech BA, Xiang MA, Linser PJ, Price DA, Van Ekeris L, Becnel JJ, Harvey WR (2012) High affinity 3H-phenylalanine uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AeBBMVw). J Insect Physiol 58:580–589CrossRefGoogle Scholar
  66. Sumner J-P, Dow JAT, Earley FGP, Klein U, Dieter J, Wieczorek H (1995) Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits. J Biol Chem 270:5649–5653CrossRefGoogle Scholar
  67. 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
  68. 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
  69. Turner RJ, Moran A (1982) Stoichiometric studies of the renal outer cortical brush border membrane D-glucose transporter. J Membr Biol 67:73–80CrossRefGoogle Scholar
  70. Volkmann A, Peters W (1989) Investigations on the midgut caeca of mosquito larvae-I. Fine structure. Tissue Cell 21:243–251CrossRefGoogle Scholar
  71. 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
  72. Weber WM (1999) Endogenous ion channels in oocytes of xenopus laevis: recent developments. J Membr Biol 170:1–12CrossRefGoogle Scholar
  73. Wieczorek H, Brown D, Grinstein S, Ehrenfeld J, Harvey WR (1999) Animal plasma membrane energization by proton-motive V-ATPases. Bioessays 21:637–648CrossRefGoogle Scholar
  74. 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
  75. Wigglesworth V (1972) Principles of insect physiology. Chapman Hall, LondonCrossRefGoogle Scholar
  76. 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
  77. Woolf B (1929) Some enzymes in B. coli communis which act on fumaric acid. Biochim 23:472–482CrossRefGoogle Scholar
  78. Xiang M, Feng M, Muend S, Rao R (2007) A human Na+/H+ antiporter sharing evolutionary origins with bacterial NhaA may be a candidate gene for essential hypertension. Proc Natl Acad Sci USA 104:18677–18681CrossRefGoogle Scholar
  79. Zhang R, Hua G, Andacht TM, Adang MJ (2008) A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae. Biochemistry 47:11263–11272CrossRefGoogle Scholar
  80. 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

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Authors and Affiliations

  1. 1.Saint James School of MedicineAnguilla BWIAnguilla
  2. 2.Department of Physiology and Functional GenomicsUniversity of FloridaGainesvilleUSA
  3. 3.Winter ParkUSA

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