Temperature and ionic strength dependence of quinacrine binding and quinacrine displacement elicited by high concentrations of agonists on the nicotinic acetylcholine receptor

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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 (K(d)) of quinacrine at the temperature range of 4-23°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°C in the absence of NaCl and at 4°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°C) and agonists at high concentrations (measured in the temperature regime of 4-15°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°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 α-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.

Original languageEnglish
Pages (from-to)1-11
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume333
Issue number1
DOIs
StatePublished - 1 Sep 1996
Externally publishedYes

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Quinacrine
Nicotinic Receptors
Ionic strength
Osmolar Concentration
Temperature
Cholinergic Receptors
Binding Sites
Ion Channels
Electric Organ
Membranes
Lipids
Torpedo
Bungarotoxins
Cholinergic Agonists
Phencyclidine
Biochemistry
Molecules
Fluorescence Spectrometry
Fluorescence spectroscopy
Carbachol

Keywords

  • agonist self-inhibitory binding site
  • fluorescence spectroscopy
  • nicotinic acetylcholine receptor
  • quinacrine binding site
  • thermodynamic parameters
  • Torpedo native membranes

Cite this

@article{ce2917502c714936bd7d4a81d64bdad5,
title = "Temperature and ionic strength dependence of quinacrine binding and quinacrine displacement elicited by high concentrations of agonists on the nicotinic acetylcholine receptor",
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 (K(d)) of quinacrine at the temperature range of 4-23°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°C in the absence of NaCl and at 4°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°C) and agonists at high concentrations (measured in the temperature regime of 4-15°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°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 α-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.",
keywords = "agonist self-inhibitory binding site, fluorescence spectroscopy, nicotinic acetylcholine receptor, quinacrine binding site, thermodynamic parameters, Torpedo native membranes",
author = "Arias, {Hugo R.}",
year = "1996",
month = "9",
day = "1",
doi = "10.1006/abbi.1996.0357",
language = "English",
volume = "333",
pages = "1--11",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
number = "1",

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TY - JOUR

T1 - Temperature and ionic strength dependence of quinacrine binding and quinacrine displacement elicited by high concentrations of agonists on the nicotinic acetylcholine receptor

AU - Arias, Hugo R.

PY - 1996/9/1

Y1 - 1996/9/1

N2 - 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 (K(d)) of quinacrine at the temperature range of 4-23°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°C in the absence of NaCl and at 4°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°C) and agonists at high concentrations (measured in the temperature regime of 4-15°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°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 α-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.

AB - 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 (K(d)) of quinacrine at the temperature range of 4-23°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°C in the absence of NaCl and at 4°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°C) and agonists at high concentrations (measured in the temperature regime of 4-15°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°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 α-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.

KW - agonist self-inhibitory binding site

KW - fluorescence spectroscopy

KW - nicotinic acetylcholine receptor

KW - quinacrine binding site

KW - thermodynamic parameters

KW - Torpedo native membranes

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DO - 10.1006/abbi.1996.0357

M3 - Article

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