当前位置： SCI文献检索 > MOLECULAR PHARMACOLOGY期刊下所有文献 > Expression of a cloned muscarinic receptor in A9 L cells.

Expression of a cloned muscarinic receptor in A9 L cells.

Abstract：

:Using an oligonucleotide based on the sequence of a porcine brain muscarinic receptor cDNA, we recently cloned four distinct muscarinic receptors from the rat and human genomes. In the present study we transfected the rat homolog of the porcine brain muscarinic receptor cDNA into A9 L cells using a mammalian expression vector and a calcium phosphate precipitation procedure. Before transfection, A9 L cells do not bind muscarinic ligands and do not express muscarinic receptor mRNA. After transfection, A9 L cells expressed muscarinic receptor mRNA and saturable, high affinity binding sites for the muscarinic antagonists 3H-quinuclidinyl benzilate and 3H-pirenzepine. The muscarinic receptor antagonists AF DX-116 and pirenzepine displaced bound 3H-quinuclidinyl benzilate with inhibition curves suggestive of a single high affinity binding site. Competition of 3H-quinuclidinyl benzilate-labeled sites with the agonists acetylcholine and carbachol yielded broad inhibition curves, consistent with a heterogeneity of binding sites. In the presence of guanine nucleotide, the agonist inhibition curves were steeper, suggesting the presence of a single low affinity site. The effects of guanine nucleotides on agonist binding are consistent with coupling of these receptors to a guanine nucleotide-binding protein (G-protein) endogenous to A9 L cells. The electrical properties of the transfected A9 L cells were examined using the whole cell patch-clamp technique. Fifty microM acetylcholine induced a conductance which reversed in polarity at -60 mV. This conductance could be reversibly blocked by atropine. These data illustrate the utility of stable transfection of A9 L cells for the characterization of individual cloned muscarinic receptors, their G-protein coupling mechanisms, and resultant physiological responses.

journal_name

Mol Pharmacol

journal_title

Molecular pharmacology

authors

Brann MR,Buckley NJ,Jones SV,Bonner TI

subject

Has Abstract

pub_date

1987-10-01 00:00:00

pages

450-5

issue

4

eissn

0026-895X

issn

1521-0111

journal_volume

32

pub_type

杂志文章

相关文献

MOLECULAR PHARMACOLOGY文献大全