Patch clamp techniques will be used to study the gating mechanism of the acetylcholine receptor channel in cultured embryonic muscle. Many of the experiments will involve chemical modifications of the receptor in which a labile disulfide near the binding site is exploited to block ligand binding selectively. Studies of spontaneous acetylcholine receptor channel gating have shown that Alpha-bungarotoxin blocks the closed-to-open conformational transition of the channel, but a chemical modification of the binding sites does not. Chemical modification of a single binding site will therefore be used to produce receptors blocked at a single binding site, and to see how they respond to ligand. Other experiments will exploit chemical reagents that are directed against the ion channel functionality of the receptor. Since local anesthetics bind to open channels, they should more frequently enter channels that stay open longer. The local anesthetic derivative quinacrine mustard, which binds covalently to the open channel, should preferentially inactivate channels that have a higher probability of being open. An irreversible channel blocker of this type could then be used to identify populations of channels that differ with regard to how much time they are open. Subconductance states were seen with ligand activated channels, but not spontaneous openings of the acetylcholine receptor channel. Recordings of channel currents following blockade of the binding site will determine if a cholinergic ligand can produce a transition to a subconductance state by binding to a site other than the sites responsible for receptor activation. A final program of experiments explores enzymatic modification of the acetylcholine receptor. The acetylcholine receptor is a phosphoprotein and is a substrate for many protein kinases. Channel gating will be examined following treatment with enzymes that change the state of phosphorylation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Modified Research Career Development Award (K04)
Project #
5K04NS001149-04
Application #
3074967
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1986-09-01
Project End
1990-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095