Partial functional reconstitution of the cardiac muscarinic cholinergic receptor

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

Research output: Contribution to journalArticle

11 Citations (Scopus)

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
Externally publishedYes

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Cholinergic Receptors
Muscarinic Receptors
Cholinergic Agents
Membranes
Erythrocyte Membrane
Molecular Weight
Molecular weight
Quinuclidinyl Benzilate
Guanylyl Imidodiphosphate
Stereoselectivity
Digitonin
Competitive Binding
Dialysis
Electrophoresis
Sodium Dodecyl Sulfate
Membrane Proteins
Gels
Binding Sites
Pharmaceutical Preparations
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Partial functional reconstitution of the cardiac muscarinic cholinergic receptor. / Shreeve, S. M.; Roeske, William R; Venter, J. C.

In: Journal of Biological Chemistry, Vol. 259, No. 20, 1984, p. 12398-13402.

Research output: Contribution to journalArticle

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