Covalent labeling of functional states of the acetylcholine receptor

Abstract

Photoaffinity labeling of membrane‐bound nicotinic acetylcholine receptor from Torpedo marmorata electric tissue with the ion‐channel blocker [3H]TPMP+ reveals various functional states of the receptor protein if labeling is performed with ms time resolution. In the resting and in the activated state most of the label is incorporated into the α‐polypeptide chains of the receptor complex. When equilibrated with agonists and antagonists, predominantly the δ‐polypeptide chain (and to a lesser extent the β‐chain) reacts with the photolabel. Reactivity of the δ‐chain increases after exposure to cholinergic effectors with a half‐life slower than the kinetics of receptor activation or rapid desensitization. Agonists and antagonists stimulate photolabelling of the δ‐chain with different kinetics. For acetylcholine, carbamoylcholine and suberyldicholine the half‐life of the reactivity increases is 400–500 ms; for the antagonists hexamethonium, d‐tubocurarine and flaxedil it is about 10 s. The latter slow kinetics are also observed when the receptor is preequilibrated with agonists or antagonists prior to mixing with [3H]TPMP+ and starting the photoreaction. We conclude that time‐resolved photoaffinity labeling can convalently mark protein structures involved in receptor functions. Of special interest is the observation that antagonists also induce a conformational change in the receptor protein.

Dynamic Structural Investigations on the Torpedo Nicotinic Acetylcholine Receptor by Time‐Resolved Photoaffinity Labeling

“…[22,[77][78][79][80] The photosensitive diazirine [ 125 I]TID (Table 3) was the first NCB to provide evidence of agonist-induced conformational changes inside the channel at the amino-acid level. This hydrophobic probe is a restingstate inhibitor that blocks ion flux by interactions with the ion-channel walls.…”