Nicotine binding to native and substituted peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha1, alpha2, alpha3, alpha4, alpha5, and alpha7 subunits.
Structural determinants of L-[(3)H]nicotine binding to synthetic peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (K(d) approximately 1.5 microM) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (K(d) = 14-23 nM) than the muscle alpha1 peptides (K(d) = 52 nM). The following nonconservative substitutions in the alpha4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195-, P199A, P199-, and Y203A. Substitution of alpha4P199 with a leucine which is present in the alpha1 sequence decreased the affinity of the alpha4 peptide for nicotine and substitution of alpha1L199 with a proline (alpha4) or a glutamine (alpha3) increased the affinity of the alpha1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the alpha4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different alpha subunits.
Lentz
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Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA. thomas.lentz@yale.edu
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006841:Hydrocarbons, Aromatic / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009538:Nicotine / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014101:Torpedo
D006841:Hydrocarbons, Aromatic; D010455:Peptides; D011978:Receptors, Nicotinic; D009538:Nicotine