A Perturbed pK_a at the Binding Site of the Nicotinic Acetylcholine Receptor: Implications for Nicotine Binding

Abstract

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at α149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pK_a of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pK_a in solution (∼9.3). This local pK_a perturbation has substantial implications for pharmacological research on the nAChR:  of the tertiary agonists considered, noracetylcholine experiences this pK_a perturbation, while nicotine does not.