Agonist-induced displacement of quinacrine from its binding site on the nicotinic acetylcholine receptor: plausible agonist membrane partitioning mechanism

Abstract

It was previously demonstrated that high concentrations of cholinergic agonists such as acetylcholine (ACh), carbamylcholine (CCh), suberyldicholine (SubCh) and spin-labelled acetylcholine (SL-ACh) displaced quinacrine from its high-affinity binding site located at the lipid-protein interface of the nicotinic acetylcholine receptor (AChR) (Arias, H. R. and Johnson, D. A. (1995) Biochemistry, 34, 1589-1595). In order to account for the agonist self-inhibitory binding site which overlaps, at least partially, with the quinacrine binding site, we determined the partition coefficient (K_p) of these agonists relative to the local anaesthetic tetracaine in AChR native membranes from Torpedo californica electric organ by examining (1) the ability of tetracaine and SL-ACh to quench membrane-partitioned 1-pyrenedecanoic acid (C₁₀-Py) monomer fluorescence, and (2) the ability of ACh, CCh and SubCh to induce an increase in the excimer/monomer ratio of C₁₀-Py-labelled AChR membrane fluorescence. To further assess the differences in agonist accessibility to the quinacrine binding site, we calculated the agonist concentration in the lipid membrane (C_M) at an external agonist concentration high enough to inhibit 50% of quinacrine binding (IC₅₀), which in turn was obtained by agonist back titration of AChR-bound quinacrine. initial experiments established that high agonist concentrations do not affect either transmembrane proton concentration equilibria (pH) of AChR membrane suspension or AChR-bound quinacrine fluorescence spectra. The agonist membrane partitioning experiments indicated relatively small (≤20) K_p values relative to tetracaine. These values follow the order: SL-ACh > SubCh s< CCh - ACh. A direct correlation was observed between K_p and the apparent inhibition constant (K_i) for agonists to displace AChR-bound quinacrine. Particularly, agonists with high K_ps such as SL-ACh and SubCh showed low K_i values, and this relationship was opposite for CCh and ACh. The calculated C_M values indicated significant (between 7 and 54 mM) agonist accessibility to lipid membrane. By themselves, these results support the conjecture that agonist self-inhibition seems to be mediated by the quinacrine binding site via a membrane approach mechanism. The existence of an agonist self-inhibitory binding site not located in the channel lumen would indicate an allosteric mechanism of ion channel inhibition; however, we can not discard that the process of agonist self-inhibition can also be mediated by a steric blockage of the ion channel.

Key Words:

• Nicotinic acetylcholine receptor
• Torpedo native membranes
• fluorescence spectroscopy
• non-competitive inhibitor binding
• agonist inhibitory binding site
• agonist partition coefficient