Biochemical Characterization of a Novel Channel-Activating Site on Nicotinic Acetylcholine Receptors

Abstract

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine and several structurally related compounds with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles, direct binding studies and photoaffinity labeling. (-)Physostigmine acts as a channel-activating ligand at low concentrations and as a direct channel blocker at elevated concentrations. Channel activation is not inhibited by desensitizing concentrations of ACh or ACh-competitive ligands (including alpha-bungarotoxin and D-tubocurarine) but is inhibited by antibody FK1 and several other compounds. From photoaffinity labeling using tritiated physostigmine and mapping of the epitope for the Phy-competitive antibody FK1, the binding site for physostigmine is located within the alpha-subunit of the Torpedo nAChR and is distinct from the acetylcholine binding site. Our data suggest a second pathway of nAChR channel activation that may function physiologically as an allosteric control of receptor activity.

The Pharmacology of Physostigmine

“…Photoaffinity labeling of nicotinic acetylcholine receptors by (+) physostigmine has revealed a pharmacologically distinct drug binding site carried on the same subunit as that for acetylcholine (246). The presence of a specific binding site was also reported by Schrattenholtz et al (228) who characterized a high affinity binding site, K D = 35 nM, for the competitive physostigmine analog 1-methylphysostigmine.…”

“…Physostigmine exerts stimulating, desensitizing, and blocking actions at nicotinic receptors in addition to its AChE-inhibitory actions at the neuromuscular junction. The agonist effects of physostigmine were observed as early as 1977 (137) and confirmed a number of times (11,202,203,228,233,234). Further support for these actions additional to AChE inhibition, stems from observations that (+) physostigmine, which is virtually devoid of AChE-inhibitory activity, is an agonist at nicotinic acetylcholine receptors (11).…”

Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels

“…Channel activation is not affected by several competitive antagonists (including oz bungarotoxin and d-tubocurarine) but is blocked by the open channel blocker dibucaine and prevented by the antibody FK1 (Okonjo, Kuhlmann & Maelicke, 1991). Benzoquinonium, a competitive antagonist of the ACh site exhibits effects that are similar to those of physostigmine (Schrattenholz et al, 1993). Photolabeling studies revealed that the binding site for physostigmine is located in the vicinity of loops A and B; the labeled residue on Torpedo californica a subunit is Lys 125 (Tano et al, 1992) which is conserved on most o~ subunits but absent on non a subunits.…”

Cholinergic Modulation by Opioid Receptor Ligands: Potential Application to Alzheimer's Disease

“…One of the first target-based models [79] used the DOCK program to bind physostigmine into a defined region located within 14Å of αLys122 residue of ACh Binding Protein (AChBP), which corresponds to the αLys125 of Torpedo nAChR identified from early photoaffinity labeling experiments [80–81] as being in or near the putative nAChR allosteric region. This study identified a hydrophobic binding site located between P-sheets B2, B5, and B6 of the α subunit and β-sheets B5 and B6 of the β subunit.…”

“…Site 3 is located close to the L5 loop and β7 and β2 sheets (β/− subunit), near the bottom of the ligand binding domain, and is poorly populated relative to sites 1 and 2. Although this third putative site is closest to that identified from photoaffinity labeling of physostigmine [80], these computational docking studies raise questions about the methodology used in those early reports. Common among these putative binding sites is a salt bridge observed between the protonated basic amine of the ligand with an anionic Asp or Glu residue contained in the nAChR.…”