Amplification of the rat m2 muscarinic receptor gene by the polymerase chain reaction: Functional expression of the M sub 2 muscarinic receptor

Journal Article · Mon Jan 01 00:00:00 EST 1990 · Life Sciences; (USA)

DOI:https://doi.org/10.1016/0024-3205(90)90472-4· OSTI ID:6254400

Lai, J; Bloom, J W; Yamamura, H I; Roeske, W R ^[1]

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1. Univ. of Arizona Health Sciences Center, Tucson (USA)

A selective amplification of the coding sequence of the rat M{sub 2} 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, pH{beta}APr-1-neo, resulted in high level expression of specific ({sup 3}H)(-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of ({sup 3}H)(-)MQNB binding with a K{sub d} value of 265 pM and a B{sub max} value of 411{plus minus}50 fmol/10{sup 6} 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 ({sup 3}H)(-)MQNB binding in these cells conformed to the characteristics of an M{sub 2} type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a K{sub i} value of 2.0 {mu}M. 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 M{sub 2} type muscarinic receptor.

• OSTI ID: 6254400
• Journal Information: Life Sciences; (USA), Vol. 47:12; ISSN 0024-3205
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PARASYMPATHOMIMETICS
RECEPTORS
GENE AMPLIFICATION
AFFINITY
BIOCHEMICAL REACTION KINETICS
CLONING
FIBROBLASTS
GENES
MICE
PARASYMPATHOLYTICS
PHOSPHOLIPIDS
POLYMERASES
RATS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMAL CELLS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
CONNECTIVE TISSUE CELLS
DRUGS
ENZYMES
ESTERS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LIPIDS
MAMMALS
MEMBRANE PROTEINS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
REACTION KINETICS
RODENTS
SOMATIC CELLS
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques