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Thermal stability ofTorpedo californica acetylcholine receptor in a cholesterol lipid environment

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Abstract

Controlled heating of acetylcholine receptor (AChR) vesicles inactivates the α-bungarotoxin (α-Bgtx) binding sites with a T50 (temperature at which 50% of the initial capacity to bind α-Bgtx remains) of 60±0.2°C. The same value was obtained for receptor reconstituted in lipid vesicles fromTorpedo electroplax where the % mol composition of cholesterol to phospholipid was 30. However, when the reconstitution was carried out in dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA) vesicles (3:1 molar ratio), T50 of the curves decreased to 56±0.2° C and no carbamylcholine stimulated22Na+ flux was detected. Inclusion of cholesterol in the DOPC-DOPA vesicles increased the toxin binding site stability. The maximal T50 of the toxin binding curves was 63±0.1° C when the % mol cholesterol/mol DOPC:DOPA in the vesicles was 33. Under these conditions AChR was able to translocate ions, a property that was lost upon heating at 46° C. Preincubation of AChR in the presence of d-tubocurarine, tetracaine or procaine did not affect T50 values of toxin binding. However, a slight increment in thermal stability was found when the receptor was preincubated in the presence of carbamylcholine. The results show that cholesterol requirements for protecting against thermal inactivation of toxin binding and ion gating properties are different and the carbamylcholine-bound receptor may have a different conformation.

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Abbreviations

AChR:

acetylcholine receptor

α-Bgtx:

α-Bungarotoxin

DOPC:

dioleoylphosphatidylcholine

DOPA:

dioleoylphosphatidic acid

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  1. Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, 64110, Kansas City, Missouri, USA

    Bernardo Perez-Ramirez

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  1. Bernardo Perez-Ramirez
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Perez-Ramirez, B. Thermal stability ofTorpedo californica acetylcholine receptor in a cholesterol lipid environment. Mol Cell Biochem 132, 91–99 (1994). https://doi.org/10.1007/BF00926917

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  • DOI: https://doi.org/10.1007/BF00926917

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