AGONIST-INDUCED DISPLACEMENT OF QUINACRINE FROM ITS BINDING-SITE ON THE NICOTINIC ACETYLCHOLINE-RECEPTOR - PLAUSIBLE AGONIST MEMBRANE PARTITIONING MECHANISM

It was previously demonstrated that high concentrations of cholinergic agonists such as acetylcholine (ACh), carbamylcholine (CCh), suberyld icholine (SubCh) and spin-labelled acetylcholine (SL-ACh) displaced qu inacrine from its high-affinity binding site located at the lipid-prot ein 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 loca l anaesthetic tetracaine in AChR native membranes from Torpedo califor nica electric organ by examining (1) the ability of tetracaine and SL- ACh to quench membrane-partitioned 1-pyrenedecanoic acid (C-10-Py) mon omer fluorescence, and (2) the ability of ACh, CCh and SubCh to induce an increase in the excimer/monomer ratio of C-10-Py-labelled AChR mem brane fluorescence. To further assess the differences in agonist acces sibility to the quinacrine binding site, we calculated the agonist con centration in the lipid membrane (C-M) at an external agonist concentr ation high enough to inhibit 50% of quinacrine binding (IC50), which i n 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) o f AChR membrane suspension or AChR-bound quinacrine fluorescence spect ra. The agonist membrane partitioning experiments indicated relatively small (less than or equal to 20) K-p values relative to tetracaine. T hese values follow the order: SL-ACh>SubCh much greater than CCh simil ar to ACh. A direct correlation was observed between K-p and the appar ent inhibition constant (K-i) for agonists to displace AChR-bound quin acrine. Particularly, agonists with high K(p)s such as SL-ACh and SubC h showed low K-i values, and this relationship was opposite for CCh an d ACh. The calculated C-M values indicated significant (between 7 and 54 mM) agonist accessibility to lipid membrane. By themselves, these r esults support the conjecture that agonist self-inhibition seems to be mediated by the quinacrine binding site via a membrane approach mecha nism. The existence of an agonist self-inhibitory binding site not loc ated in the channel lumen would indicate an allosteric mechanism of io n channel inhibition; however, we can not discard that the process of agonist self-inhibition can also mediated by a steric blockage of the ion channel.