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  • Title: Temperature and ionic strength dependence of quinacrine binding and quinacrine displacement elicited by high concentrations of agonists on the nicotinic acetylcholine receptor.
    Author: Arias HR.
    Journal: Arch Biochem Biophys; 1996 Sep 01; 333(1):1-11. PubMed ID: 8806747.
    Abstract:
    This paper displays an attempt to elucidate the inhibitory mechanism of the nicotinic acetylcholine receptor (AChR) by spectroscopic means. Specifically, quantitative fluorescence spectroscopy was used to characterize: (1) the mechanism of quinacrine binding to its high-affinity noncompetitive inhibitor site located at the lipid-protein interface of the AChR and (2) the process by which agonists at high concentrations sterically compete for the quinacrine locus. For the first purpose, we study the temperature and ionic strength dependence of quinacrine binding by measuring the apparent dissociation constant (Kd) of quinacrine at the temperature range of 4-23 degrees C and in the sodium chloride (NaCl) concentration order of 0-250 mM. For the second objective, AChR native membranes from Torpedo californica electric organ suspended in buffer 10 mM sodium phosphate, pH 7.4, were preincubated with quinacrine for 2 h in the presence or in the absence of phencyclidine (PCP). Then, the PCP-sensitive quinacrine fluorescence was monitored while high concentrations of cholinergic agonists such as suberyldicholine, acetylcholine (ACh), or carbamylcholine were added to the suspension. By repeating these agonist back titrations at 4, 9, and 15 degrees C in the absence of NaCl and at 4 degrees C in the presence of 100 mM NaCl, we determined the temperature and ionic strength dependence of agonist binding to the quinacrine domain. These experiments suggest that the binding of both quinacrine (measured in the temperature range from 15 to 23 degrees C) and agonists at high concentrations (measured in the temperature regime of 4-15 degrees C) are enthalpy-driven processes, albeit that quinacrine binding is exothermic and agonist binding is endothermic. One plausible model to explain our results is that the quinacrine molecule needs first to be sterically well oriented to further enter into its binding site located in a crevice at the lipid-protein interface, whereas agonist molecules do not. Additionally, a relatively minimal electrostatic component is present in the quinacrine locus. Interestingly, the agonist inhibition constant values determined at 4 degrees C in the presence of 100 mM NaCl showed an exact correlation (slope = 1.03) with the reported concentration values of agonist that inhibit 50% of the maximum 86Rb+ efflux from AChR native vesicles in a 10-s assay with 80-85% of the alpha-bungarotoxin AChR sites occupied at zero membrane potential [S. A. Forman, L. L. Firestone, and K. W. Miller (1987) Biochemistry 26, 2807-2814]. This interdependence strongly supports the existence of a structural relationship between the agonist self-inhibitory binding site and the quinacrine locus. Although there exist evidence indicating that the process of agonist self-inhibition is mediated by a steric blockage of the ion channel, the occurrence of an agonist self-inhibitory binding site not located in the lumen channel indicates an allosteric mechanism for ion channel inhibition.
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