A covalently bound photoisomerizable agonist: comparison with reversibly bound agonists at Electrophorus electroplaques.

Abstract

After disulphide bonds are reduced with dithiothreitol, trans-3-(a-bromomethyl)-3'-[a-(t rimethylammonium) 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 btM carbachol. Voltage-jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 #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" by QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3',bis-[a-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis ~ trans photoisomerizations and decreased by trans --* dsphotoisomerizations. As with Bis-Q, light-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered cis-QBr have a channel duration severalfold briefer than with the tethered trans isomer. By comparing the agonist-induced conductance with the cis/trans 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. J. GEN. PHYSIOL. ~) The Rockefeller University Press

Flipping the Photoswitch: Ion Channels Under Light Control

“…Bromomethyl-AB-QA (QBr) attached to a native Cys of endogenous nAChRs and enabled selective channel opening in its trans-isomer (Bartels et al 1971). QBr was subsequently applied to study ion channel activation kinetics in Electrophorus electroplaques and rat myoballs (Chabala and Lester 1986;Lester et al 1980). Unlike QBr, which was not targeted by genetic manipulation, recent PTLs were designed to act as an agonist (Mal-ABacylcholine (MAACh)) or antagonist (Mal-AB-homocholine (MAHoCh)) on genetically engineered nAChRs ( Figure 5) .…”

“…One central motivation for this work was found in the recognition that light can be precisely controlled in space and time and offers non-invasive 'remote' control in transparent matrices. Inspired by classic work dating back as far as to the 1970s (Bartels et al 1971; Lester et al 1980;Bieth et al 1969;Deal et al 1969), photochromes were recently introduced in the ion channel field to contribute these experimental advantages to our current research. Researchers began to exploit photochromes with the help of molecular, chemical and genetic engineering in the fields of photopharmacology and optochemical genetics.…”

Activation of acetylcholine receptor channels by covalently bound agonists in cultured rat myoballs.

“…We have therefore combined whole-cell and single-channel measurements on cultured rat myoballs in order to extend earlier experiments with tethered agonists at the ACh receptor of frog muscle (Cox et al 1979) and ofElectrophorus electroplaques (Lester et al 1980). As found previously, the ACh channel can be activated by a single tethered agonist, although other channel properties, such as the conductance, kinetics, and voltage dependence, are very similar to those seen with reversibly bound agonists.…”

“…Unreacted ligand molecules are then washed out of the bathing solution, but the covalently bound (' tethered ') molecules still produce either persistent activation or inactivation, depending on the structure of the alkylating agent. Bartels, Wassermann & Erlanger (1971) introduced a photoisomerizable tethered agonist, QBr, which has been studied in greater detail at Electrophorus electroplaques (Lester, Krouse, Nass, Wassermann & Erlanger, 1980). Only trans-QBr is an agonist; and the photoisomerizations can be performed while the molecule is tethered to receptors, with the expected relaxations of the agonist-induced conductance.…”

“…The tethered agonist is present at a very high effective local concentration: Cox, Kawai, Karlin & Brandt (1979) estimated -1 M. If a reversibly TETHERED NICOTINIC AGONISTS AT RAT MYOBALLS bound agonist molecule were studied at such concentrations, channel block by the agonist itself would intolerably complicate the current records. In addition, by comparing the fractional conductance with the fraction of active (trans) tethered QBr, it has been shown that the open state of the receptor is controlled by the configuration of a single tethered QBr molecule (Lester et al 1980). This contrasts with the usual situation for reversibly bound agonists, in which the open state of the receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule.…”