CHARACTERIZATION OF A NICOTINIC ACETYLCHOLINE-RECEPTOR FROM THE INSECT MANDUCA-SEXTA

Manduca sexta is a nicotine-insensitive insect, the larval form of whi ch feeds on tobacco. It has been postulated that its nicotine insensit ivity may reflect the presence of a modified nicotinic acetylcholine r eceptor whose or subunits lack the amino acid residues necessary for b inding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [I-125]alpha-Bungarotoxin bound s pecifically to both larval and adult membranes, with K-d values of 7.6 and 6.5 nM and B-max values of 119 and 815 fmol/mg protein, respectiv ely. The pharmacological profile of [I-125]alpha-bungarotoxin binding was similar in both tissues. In particular, nicotine (K-i values: 1.6 mu M and 2 mu M for larvae and adults, respectively) competed with an affinity similar to that found for nicotine-sensitive insects. No alph a-bungarotoxin-insensitive binding sites labelled by [H-3]epibatidine could be detected. Using the alpha-like subunit from the locust Schist ocerca gregaria to probe two cDNA libraries, and by inverse PCR on cir cularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative n icotinic subunit from Manduca sexta (MARA1). No other alpha-subunit cD NAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an alpha-like subunit capable of binding alpha-bungarotoxin, and it retai ns all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta i s the result of a nicotinic receptor with diminished nicotine binding.