Partial functional reconstitution of the cardiac muscarinic cholinergic receptor

S. M. Shreeve, W. R. Roeske, J. C. Venter

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a K(d) (dissociation constant) equal to 48 ± 4 pM and a B(max) (maximal density of binding sites) equal to 50 ± 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 ± 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.

Original languageEnglish (US)
Pages (from-to)12398-13402
Number of pages1005
JournalJournal of Biological Chemistry
Volume259
Issue number20
StatePublished - 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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