Rates and Equilibria for a Photoisomerizable Antagonist at the Acetylcholine Receptor of Electrophorus Electroplaques

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[α-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 µM for the cis and trans isomers, respectively. Flash-induced trans → cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately ~ 10^8 M^(-1) s^(-1) and 60 s^(-1) at 20ºC; the Q_(10) is 3. Flash-induced cis → trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3-(alpha-bromomethyl)-3’-[alpha-(trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 mu M carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules

Light-activated drug confirms a mechanism of ion channel blockade

A variety of mechanisms underlie the pharmacological blockade of membrane excitability. Some drugs seem to reduce the frequency at which ion channels open; a good example is the effect of curare on acetylcholine receptor channels at normal resting potentials. Another sort of mechanism may account for the action of many local anaesthetics and related drugs containing charged ammonium groups. It is postulated that such molecules block transmembrane currents as they bind to sites within open ion channels, much like a plug in a drain, with the important difference that the events occur on a millisecond time scale. This model, which we shall call ‘open-channel blockade', was first applied to the effect of internal tetraethylammonium ions on K⁺ channels in squid axon and more recently to similar actions of local anaesthetics on acetylcholine receptor channels and on electrically excitable Na⁺ channels. (Curare seems to exert an additional open-channel blockade at high negative potentials.) The concept of open-channel blockade would receive direct experimental support from the demonstration that the blockade is exerted even if the blocking molecule is not bound to the channel (or indeed is not present at all) until after the channel opens. Such a demonstration is made possible by a drug that (1) blocks acetylcholine receptor channels in Electrophorus electroplaque, and (2) is created, in less than a millisecond, by a flash of light

Light-activated drug confirms a mechanism of ion channel blockade

A variety of mechanisms underlie the pharmacological blockade of membrane excitability. Some drugs seem to reduce the frequency at which ion channels open; a good example is the effect of curare on acetylcholine receptor channels at normal resting potentials. Another sort of mechanism may account for the action of many local anaesthetics and related drugs containing charged ammonium groups. It is postulated that such molecules block transmembrane currents as they bind to sites within open ion channels, much like a plug in a drain, with the important difference that the events occur on a millisecond time scale. This model, which we shall call ‘open-channel blockade', was first applied to the effect of internal tetraethylammonium ions on K⁺ channels in squid axon and more recently to similar actions of local anaesthetics on acetylcholine receptor channels and on electrically excitable Na⁺ channels. (Curare seems to exert an additional open-channel blockade at high negative potentials.) The concept of open-channel blockade would receive direct experimental support from the demonstration that the blockade is exerted even if the blocking molecule is not bound to the channel (or indeed is not present at all) until after the channel opens. Such a demonstration is made possible by a drug that (1) blocks acetylcholine receptor channels in Electrophorus electroplaque, and (2) is created, in less than a millisecond, by a flash of light