A photoisomerizable muscarinic antagonist: Studies of Binding and of Conductance Relaxations in Frog Heart

Abstract

These experiments employ the photoisomerizable compound, 3,3'- bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans-Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 AM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltageclamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (<1 ms duration) from a xenon flashlamp. Trans → cis and cis → trans photoisomerizations cause small (<20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23°C, k is ~3 s-1 and n is 2. Neither knor n is affected when: (a) [Bis-Q] is varied between 5 and 100 µM; (b) [carbachol] is varied between I and 50 µM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-,β-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30°C, k increases with temperature; the Qto is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence oftwo (or more) steps with a high activation energy. © 1982, Rockefeller University Press., All rights reserved.