Photoaffinity labeling of Torpedo acetylcholine receptor by physostigmine

Abstract

The plant alkaloid physostigmine, an established anti-cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorutu electrocytes [Okonjo, K. O., Kuhlmann, J. & Maelicke, A. (1991) Eur. J. Biochem. 200, Pharmacological studies of physostigmine-induced ion flux into nicotinic-acetylcholine-receptorrich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl-(n)-'HI( -)physostigmine, the physostigmine-binding site is located within the same subunit (a polypeptide) of the receptor as the acetylcholine-binding site. Using a variety of proteolytic cleavage conditions for the purified a polypeptide, several ['Hlphysostigmine-labeled peptides were isolated and sequenced. From the radioactivity released in the course of the Edman degradations of the labeled peptides, it was found that the label was associated in all cases with Lys125. These results identify a novel ligand-binding site for the Torpedo nicotinic acetylcholine receptor that is different in location from binding sites identified previously for acetylcholine, its established agonists and antagonists, and di.rect channel blockers.(-)Physostigmine (eserine), the major alkaloid of the calabar bean Physostigma venenosum Balfour [l], is a slowly reversible inhibitor of acetylcholine esterase (AChE), acting by carbamoylation of the active serine residue within the 'esteratic site' of the enzyme [2, 31. This ancient drug [4] continues to be widely used as a diagnostic aid, as an analgesic, in the recovery from anaesthesia, in the treatment of some types of psychosis, for the treatment of alcohol intoxication and glaucoma, and, most recently, for the treatment of acute brain hypoxia IS] and Alzheimer's disease [6, 71. Since physostigmine readily penetrates the bloodhrain barrier, it mainly acts in the braidcentral nervous system. In spite of its wide-spread medical applications and long use in research, however, many of its actions are not yet thoroughly understood, including those at cholinergic and serotonergic synapses [8-141. Evidence has been assembled over the past few years suggesting that, in addition to acting as an inhibitor of AChE, the drug directly interacts with both muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors [ 10-141.Studying frog muscle nAChR, we have recently demonstrated that physostigmine, below 1 pM, induces single-channel currents with amplitudes and open-time characteristics typical for the nAChR channel [12,. At higher con- [20, 211. Surprisingly, the direct action of physostigmine at the Torpedo nAChR was unaffected by the presence of saturating concentrations of acetylcholine antagonists, including a bungarotoxin and D-tubocurarine 1201, or when nAChR was desensitized by elevated concentrations of acetylcholine [21]. In addition, we established that two monoclonal antibodies raised against nAChR could competitively inhibit the binding of physostigmi...

Potentiation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

“…Allosteric potentiators, such as galantamine and physostigmine, are thought to interact with a site located in the nAChR extracellular domain (39)(40)(41), perhaps analogous to the site for potentiation of GABA A receptors by benzodiazepines (41). It appears, therefore, that nAChRs and other Cys-loop receptors can be modulated by allosteric effectors acting at multiple independent sites.…”

Unconventional ligands and modulators of nicotinic receptors

“…Subsequently, physostigmine was found to activate Torpedo nAChRs as well as neuronal nAChRs expressed natively in hippocampal neurons or ectopically in fibroblasts (Okonjo et al, 1991;Pereira et al, 1993Pereira et al, , 1994. Physostigmine-induced activation of nAChRs was insensitive to blockade by competitive nAChR antagonists and could be observed when the receptors were desensitized by high agonist concentrations (Okonjo et al, 1991;Pereira et al, 1993;Schrattenholz et al, 1993). The monoclonal antibody raised against the Torpedo nAChR ␣ subunit and referred to as FK1 is the only known antagonist of the nicotinic agonistic action of physostigmine.…”

“…3(A); Schrattenholz et al, 1993]. Photoaffinity labeling studies carried out using [ 3 H]physostigmine and mapping of the epitope of the monoclonal antibody FK1 revealed that the region flanking the amino acid Lys-125 on the nAChR ␣ subunit contains essential elements of the physostigminebinding site and is highly conserved among different ␣ subunits and across species (Pereira et al, 1993;Schrattenholz et al, 1993;Schroder et al, 1994).…”

Expression and Renaturation of the N-terminal Extracellular Domain of Torpedo Nicotinic Acetylcholine Receptor α-Subunit

“…Materials-nAChR-rich Torpedo membrane fragments (3 mg/ml; 5 nmol of ACh sites/mg) were obtained by homogenization of Torpedo marmorata electric organ, followed by several centrifugation steps and alkaline treatment as described (11). N-␣- [propionyl-3 H]bungarotoxin (54 -68 Ci/mmol) was obtained from Amersham Pharmacia Biotech.…”

“…These studies have elucidated the overall dimensions of the receptor protein (3), its position with respect to the surrounding lipid bilayer, the locations of functional domains and amino acid residues belonging to the integral ion channel (4,5), its gating structures (6,7), and the binding sites for several classes of ligands (1,2,(7)(8)(9)(10)(11)(12). However, a high resolution three-dimensional structure of the nAChR or any other neuroreceptor is still missing.…”