Photochromic Activators of the Acetylcholine Receptor

Abstract

Two photochromic activators of the electrogenic membrane of the electroplax of Electrophorus electricus are described. monium)methyljazobenzene dibromide (Bis-Q), one of the most potent ever reported, is active at concentrations of less than 10-7 M. Its cis isomer, which is obtained from the trans by exposure to light of 330 nm, is practically devoid of activity. Photoregulation of the potential of the membrane takes place in the presence of Bis-Q, presumably because of the conversion of the active trans isomer to the inactive cis isomer in the single-cell electroplax system.The second activator, 3-(a-bromomethyl)-3'-[a-(trimethylammonium)methyllazobenzene bromide (QBr) can be covalently attached to the electroplax membrane after reduction of the membrane with dithiothreitol. Activation of the membrane is induced by the covalently linked reagent. Its cis isomer, obtained from the trans by exposure to light of 330 nm, is, like cis-Bis-Q, of very low activity. Both isomers of Bis-Q are equally active as inhibitors of acetylcholinesterase, 50% inhibition occurring at a concentration of 10-5 M. The possibility of using trans-Bis-Q and trans-QBr to characterize and isolate the receptor protein is discussed.Systems in which photoregulation could be studied at the molecular level were described in previous papers. In these systems, photochromic azo derivatives were used as effector molecules to regulate the activities of chymotrypsin (1) and acetylcholinesterase (2, 3) and to photoregulate the potential of the excitable membrane of the monocellular electroplax preparation (4). Photoregulation was achieved by exploiting differences between the biochemical activities of the cis and trans isomers of the photochromic compounds, the relative concentrations of which were influenced by the wavelength of light to which the solution was exposed [or light vs. darkness, in one case (3) ].Light-induced changes in potential of the electroplax membrane may be considered as a Mtodel for the process of vision, in which the cis to trans isomerization of retinal is the first step in the initiation of a neural impulse. In the latter case, however, as well as in the phytochrome system of plants (5), the photochromic substances are located intracellularly, making for a highly efficient process. It thus appeared of interest to prepare a light-sensitive ligand that would form a covalent bond with the receptor protein of the electroplax. A compound with the desired properties was prepared: 3-(a-bromomethyl)-3'-[a-(trimethylammonium)methyl]azobenzene (QBr). Also synthesized was the closely related 3,3'-bis[a-(trimethylammonium)methyl]azobenzene (Bis-Q). the trans isomer of which was found to be a potent receptor activator, one of the most potent thus far described.The high affinity and specificity of Bis-Q may make it a useful reagent for the characterization, isolation, and purification of the receptor protein. Some experiments with the two azo compounds are presented in this paper. METHODSPreparation of 3,3'-bis(a-bromomethyl)azobenz...

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

“…PTLs comprise a modular class of genetically-targeted photochemical tools first conceived in the 1970s by Eranger and Lester for acetylcholine receptors (19,20). PTLs have since been developed for a variety of other receptors and channels (2).…”

Flipping the Photoswitch: Ion Channels Under Light Control

“…These molecules were mono-or bifunctional with a quaternary ammonium (QA) and were applied to control nicotinic acetylcholine receptors (nAChRs) and acetylcholine esterase to study kinetics of ion channel activation and membrane potential shifts in excitable tissue (Bartels et al 1971;Bieth et al 1969;Deal et al 1969;Chabala et al 1985;Lester et al 1979;Nargeot et al 1982). Despite these early successful examples for the optical control of ion channel function, further design and application of PCLs was not revisited until nearly three decades later.…”

“…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.…”

Chemical Events in Conducting and Synaptic Membrances during Electrical Activity

“…Just as for the enzyme, the electric tissue offers a uniquely favorable material for isolation of the receptor protein, especially in combination with the information obtained about some of its characteristics. In the last few years, several groups have devoted great efforts to isolate and characterize the receptor protein from electric tissue, applying a variety of procedures (29,(31)(32)(33)(34)(35)(36)(37)(38)(39). A few aspects pertinent to this presentation may be mentioned.…”