Single Channel Studies of Acetylcholine Receptors Covalently Alkylated with Acetylcholine

Abstract

Ever since the pioneering work of del Castillo and Katz (1957), a close correlation between acetylcholine receptor (AChR) occupancy by transmitter (or agonist) and the mean open time of the transmitter-activated channel has been assumed. Supporting lines of evidence are (i) the dependency of the mean open time on agonist structure (Katz and Miledi, 1973; Anderson and Stevens, 1973; Neher and Stevens, 1977), and (ii) the apparent “persistent” channel activation upon affinity-labeling of the dithiothreitol (DTT)-treated AChR with bromoacetylcholine (BrACh, Bartels and Karlin, 1966; Bartels et al., 1976). For several reasons, however, the above assumption has never been conclusively established: (i) For technical reasons, there do not exist reliable data on the dissociation rate constants of representative nicotinic agonists and the active state of the AChR. As a consequence, the assumed correlation between mean open time and agonist occupancy has not yet been tested quantitatively. (ii) Persistent depolarization after covalent labeling with agonist has only been observed in unclamped membrane voltage studies with elecnophonus electroplaque cells (Bartels et al., 1976). Comparable studies with voltage-clamped frog muscle (Ivens, unpublished result) provided initial indication that covalent agonist coupling only results in prolonged but not persistent channel activation. (iii) Studies with the “tethered agonist” QBr (Lester et al., 1980) indicate that at least agonists covalently linked via a long bridge to the AChR, do not irreversibly activate the receptor channel.