Premium
Characterization of a nicotinic acetylcholine receptor from the insect Manduca sexta
Author(s) -
Eastham Helen M.,
Lind Robert J.,
Eastlake Jane L.,
Clarke Barry S.,
Towner Paul,
Reynolds Stuart E.,
Wolstenholme Adrian J.,
Wonnacott Susan
Publication year - 1998
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1046/j.1460-9568.1998.00095.x
Subject(s) - manduca sexta , nicotinic agonist , nicotine , epibatidine , manduca , biology , acetylcholine receptor , biochemistry , nicotinic acetylcholine receptor , complementary dna , sphingidae , microbiology and biotechnology , protein subunit , receptor , insect , gene , botany , neuroscience
Abstract Manduca sexta is a nicotine‐insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose α subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [ 125 I]α‐Bungarotoxin bound specifically to both larval and adult membranes, with K d values of 7.6 and 6.5 n m and B max values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [ 125 I]α‐bungarotoxin binding was similar in both tissues. In particular, nicotine ( K i values: 1.6 μ m and 2 μ m for larvae and adults, respectively) competed with an affinity similar to that found for nicotine‐sensitive insects. No α‐bungarotoxin‐insensitive binding sites labelled by [ 3 H]epibatidine could be detected. Using the α‐like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other α‐subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an α‐like subunit capable of binding α‐bungarotoxin, and it retains 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 is the result of a nicotinic receptor with diminished nicotine binding.