Native folding of an acetylcholine receptor α subunit expressed in the absence of other receptor subunits

P. Blount, J. P. Merlie

Research output: Contribution to journalArticle

79 Scopus citations

Abstract

An α subunit of cDNA of the mouse nicotinic acetylcholine receptor under transcriptional control of the Rous Sarcoma virus long terminal repeat was transfected into and expressed in a quail fibroblast cell line. The biosynthesis and post-translational modification of the α subunit protein made in this heterologous system have been studied using immunoprecipitation and ligand binding assays. The polypeptide is present at high steady-state levels and inserted in the correct trans-membrane orientation. However, in the absence of assembly with other subunits the α subunit is confined to an intracellular membrane compartment and is not transported to the plasma membrane. Twenty percent of the newly synthesized α subunit acquired high affinity α bungarotoxin binding in a time-dependent process within 20 min of translation. Sucrose gradient fractionation demonstrated that both the polypeptide and toxin binding forms of the α subunit have a sedimentation coefficient of 5 s suggesting the absence of stable homo-oligomers. Quantitative binding assays demonstrated that the apparent affinity and rate of association of α bungarotoxin to the unassembled α subunit are greater than for native receptor. On the other hand, the affinities for the small ligands D-tubocurarine and gallamine are 103 lower than for native receptor; no detectable binding was observed for decamethonium, hexamethonium, or carbamylcholine. Thus, the acetylcholine receptor α subunit, independent of other subunits of the receptor, acquires a mature conformation and high affinity α bungarotoxin binding when expressed in a quail fibroblast cell line.

Original languageEnglish (US)
Pages (from-to)1072-1080
Number of pages9
JournalJournal of Biological Chemistry
Volume263
Issue number2
StatePublished - Jan 1 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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