Period Covered: October 1, 1978 - September 30, 1979
Title of Project: Muscarinic Acetylcholine Receptors of Cultured Cell Lines
Names, Laboratory and Institute Affiliations, and Titles of Principal Investigations and All Other Professional Personnel
Engaged on the Project:
PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI
Other: William L. Kline, Guest Worker, LBG NHLBI
Orest Hurko, Staff Associate, LBG NHLBI
Cooperating Units (if any): None
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section on Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, MD 20205
Total Man Years: 0.25
Summary of Work: Studies on: muscarinic acetylcholine receptors focus both on ligand-binding and on defining the physical
properties of muscarinic receptors.
Major Findings: [3H]-Quinuclidinyl-benzilate (QNB) was used to study muscarinic acetylcholine receptors in NG108-15 membrane
preparations. The apparent dissociation constant of [3H]-QNB is 1 x 10^(-10) M; the average NG108-15 cell possesses 30,000
specific sites for [3H]-QNB. Activation of the receptors with acetylcholine or carbachol results in cell depolarization,
a small increase in cellular cGMP, and inhibition of adenylate cyclase. Cell depolarization and rise in cGMP levels desensitize
in 30 sec; whereas, the inhibition of adenylate cyclase does not desensitize. Scatchard analysis revealed only one homogeneous
class of [3H]-QNB binding sites; however biphasic rates of [3H]-QNB association with and dissociation from receptors were
found. Evidence was obtained for the formation of a dissociable [[3H]-QNB Receptor] complex which then is converted to a
form which dissociates only slowly. Hill coefficients of approximately 1.0 were found for receptor antagonists and approximately
0.5 for receptor activators. A sequential series of reactions were proposed to account for these observations and for the
various states of the muscarinic acetylcholine receptor that were detected.
1. Burgermeister, W., Kline, W.L., Nirenberg, M., and Witkop, B., Mol. Pharm. 14, 751-767 (1978).
2. Hurko, O. Specific [3H]-Quinuclidinyl Benzilate binding activity in digitonin-solubilized preparations for bovine brain.
Arch. Biochem. and Biophys. 190, 434-445 (1978).