Amplification of the rat m2 muscarinic receptor gene by the polymerase chain reaction: Functional expression of the M2 muscarinic receptor

Josephine Lai, John W. Bloom, Henry I. Yamamura, William R. Roeske

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pHβAPr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H](-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411±50 fmol/106 cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H](-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 μM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.

Original languageEnglish (US)
Pages (from-to)1001-1013
Number of pages13
JournalLife Sciences
Volume47
Issue number12
DOIs
StatePublished - 1990

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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