Amino acids within residues 181-200 of the nicotinic acetylcholine receptor alpha1 subunit involved in nicotine binding.

Structural determinants of L-[3H]nicotine binding to the sequence flanking Cys 192 and Cys 193 of the Torpedo acetylcholine receptor alpha1 subunit were investigated using synthetic peptides (residues 181-200) and fusion proteins (residues 166-211). Nicotine binding at a single concentration (30 nM) was compared with 71 peptides and fusion proteins in which individual amino acids at positions 181-200 were substituted. Substitution of Lys 185, Tyr 190, Cys 192, Cys 193, Thr 196, and Tyr 198 resulted in the greatest reduction in nicotine binding. Equilibrium binding of [3H]nicotine to peptide 181-200 revealed a binding component with an apparent KD of 1.2 microM. Substitution of Lys 185 (with Glu), His 186, Tyr 190, Cys 192, Cys 193, and Tyr 198 resulted in a significant reduction in affinity. Affinity was not affected significantly by substitution of Arg 182, Lys 185 (with Gly or Arg), Val 188, Tyr 189, Pro 194, Asp 195, Thr 196, and Asp 200. It is concluded that Lys 185, His 186, Tyr 190, Cys 192, Cys 193, and Tyr 198 play the greatest role in nicotine binding to residues 181-200 of the alpha1 subunit. Previous studies have implicated Tyr 190, Cys 192, Cys 193, and Tyr 198 in agonist binding to the acetylcholine receptor. These results confirm a role for these residues and also demonstrate a function for Lys 185 and His 186 in nicotine binding.