Original Publication Date
The Biophysical Journal
DOI of Original Publication
Date of Submission
Our group initiated the use of pyrene derivatives for functional and structural studies of the membrane-bound Acetylcholine Receptor (AcChR) system. Our studies have taken two approaches. We have recently used a hydrophobic derivative (pyrene-l-sulfonyl azide) that is able to bind covalently to regions of the AcChR molecule accessible from the membrane lipid (1) to monitor ligandinduced effects at those hydrophobic regions of the AcChR protein exposed to the membrane matrix (2). The structural effects induced by ligand binding are interpreted in relation to the AcChR "desensitization" process. We have also used a hydrophilic pyrene derivative (1,3,6,8-pyrene tetra sulfonate [PTSA]) to develop a fast spectroscopic method for measuring AcChR functionality, i.e., the formation of a short-lived cation channel, within the physiological time scale. The procedure is based on the fluorescence quenching of intravesicularly entrapped PTSA by externally added Tl which substitutes for the physiologically occurring Na+. Addition of cholinergic agonist to preparations of AcChR membranes containing entrapped PTSA promotes the formation of the AcChR cation channel, which allows the added Tl+ into the internal volume of the vesicles where collisional quenching between the cation and the entrapped fluorophore occurs. This Tl+ influx is spectroscopically monitored as a time-dependent decrease of the fluorescence emitted by PTSA in the millisecond time scale. In this communication, we use the latter, "stoppedflow/fluorescence quenching," procedure to examine several functional aspects of the membrane bound AcChR, including the effect of alkaline extraction of peripheral proteins from the AcChR membranes, and the effect of binding of specific anti-AcChR antibodies, which serves as a molecular model for the autoimmune disease Myasthenia gravis(4, 5)
From The Biophysical Journal, Martinez-Carrion, M., Gonzalez-Ros, J.M., Mattingly, J.R., et al., Fluorescence Probes for the Study of Acetylcholine Receptor Function, Vol. 45, Page 141. Copyright © 1984 The Biophysical Society. Published by Elsevier Inc. Reprinted with permission.
Is Part Of
VCU Biochemistry and Molecular Biology Publications