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

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 > Na) or the external fluid-membrane interface (Na < Na). 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.