The nicotinic acetylcholine receptor: Binding of nitroxide analogs of a local anesthetic and a photoactivatable analog of phosphatidylserine
Electron spin resonance was used to contrast the accessibility of tertiary and quaternary amine local anesthetics to their high affinity binding site in the desensitized Torpedo californica acetylcholine receptor (AchR). Preincubation of AchR-rich membranes with agonist resulted in a substantial reduction in the initial association of the quaternary amine local anesthetic C6SLMEI with the receptor. The time-dependent reduction in association follows a biphasic exponential function having rate constants of 0.19 min{sup {minus}1} and 0.03 min{sup {minus}1}. In contrast, agonist preincubation did not produce a comparable decrease in the association of C6SL, a tertiary amine analog, with the AchR. The results are modeled in two ways: (1) A charge gate near the channel mouth in the desensitized receptor limits access of the permanently charged cationic local anesthetic (C6SLMEI), but not for the uncharged form of the tertiary amine anesthetic C6SL. (2) A hydrophobic pathway, possibly through a corridor in the annular lipid surrounding receptor subunits, allows the uncharged form of C6SL to reach the high affinity binding site in the AchR. A photoactivatable analog of phosphatidylserine {sup 125}I 4-azido salicylic acid-phosphatidylserine ({sup 125}I ASA-PS) was use to label both Torpedo californica acetylcholine receptor-rich membranes and reconstituted AchR membranes. All four subunits of the AchR were found to incorporate label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporate {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. Eighty-one per cent of the incorporated label was localized to 11.7 and 10.1 kdal V8 cleavage fragments.
- Research Organization:
- California Univ., Santa Cruz, CA (United States)
- OSTI ID:
- 5545673
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
AFFINITY
AMINES
AMMONIUM COMPOUNDS
ANESTHETICS
ANIMALS
AQUATIC ORGANISMS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM DEPRESSANTS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTRON SPIN RESONANCE
ESTERS
FISHES
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIPIDS
MAGNETIC RESONANCE
MEMBRANE PROTEINS
MEMBRANES
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PARASYMPATHOMIMETICS
PHOSPHOLIPIDS
PROTEINS
QUATERNARY COMPOUNDS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RESONANCE
TIME DEPENDENCE
TRACER TECHNIQUES
VERTEBRATES