DETERMINANTS FOR THE ACTIVITY OF THE NEUTRAL AMINO ACID/K+ SYMPORT IN LEPIDOPTERAN LARVAL MIDGUT

Abstract

The columnar cells of lepidopteran larvae express, in their apical brush-border membrane, a class of symporters which in vivo couple the intracellularly directed amino acid and K+ fluxes. An analysis of the functional properties of the symporter for neutral amino acids along the anterior, middle and posterior regions of the larval midgut of Bombyx mori demonstrated the ability of a K+ gradient to drive leucine accumulation into brush-border membrane vesicles (BBMV) in all three preparations. However, marked differences are evident between the posterior (P) and the anterior­middle (AM) regions. In P-BBMV, much higher intravesicular accumulations were observed, Vmax was six- to eightfold higher than in AM-BBMV, a lowering of external pH (pHe) from 8.7 to 7.2 caused a tenfold increase of Km, and the absence of a potential difference (delta psi) caused a threefold decrease of Vmax. In contrast, leucine uptake in AM-BBMV was poorly sensitive to both pH and delta psi. The kinetics of leucine uptake as a function of cis K+ concentration were hyperbolic in P-BBMV and sigmoidal in AM-BBMV. More than 50 amino acids and analogues were used in inhibition experiments to characterize the amino acid binding site. Branched-chain amino acids modified on the carboxyl moiety were recognized only by the P-BBMV symporter. In AM-BBMV, substrate affinity was increased by the presence of a heterocyclic sidechain, even in the presence of a modified carboxyl- or alpha-amino group. Together, these results suggest that isoforms of the neutral amino acid/K+ symporter are present. A natural inhibitor of amino acid symport has not yet been identified. However, several lines of evidence suggest that strong interactions exist between the amino acid/K+ symporter and the receptor for the lepidopteran-specific Bacillus thuringiensis delta-endotoxins. CryIA(a) toxin, highly toxic for B. mori larvae, produced a dose-dependent inhibition of leucine uptake into both BBMV populations. The toxin was able to block the symporter in its ternary and leucine-only forms.