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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
Article
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Abstract

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.

Keywords

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

Abbreviations

AeBBMVw

Aedes aegypti brush border membrane vesicles from whole larvae

OMP

Other membrane proteins

AO

Acridine orange

HEPES

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

Tris

Tris(hydroxymethyl)aminomethane

MOPS

4-Morpholinepropanesulfonic acid

TMA

Tetramethylammonium

GC

Gastric caeca

AMG

Anterior midgut

PMG

Posterior midgut

MP

Malpighian tubules

APN2

N-Aminopeptidase/Bacillus thuringiensis israelensis (Bti) receptor

NHA

Na+/H + antiporter

NAT

Nutrient amino acid transporter

NHE

Na+/H + exchanger

VAN

V-ATPase, antiporter, nutrient amino acid transporter

Notes

Acknowledgements

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.

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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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