Desensitization of Mouse Nicotinic Acetylcholine Receptor Channels A Two-Gate Mechanism

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unligandedor diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition ( e or g ), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed. key words: single-channel • kinetics • electrophysiology i n t r o d u c t i o n Acetylcholine receptors (AChR) 1 are ion channels that open transiently after binding two agonist molecules. In the continuous presence of agonist, AChR become refractory to the stimulus and the cellular response declines. This process, called desensitization, occurs because AChR adopt liganded, stable conformations through which ions cannot permeate (reviewed by Ochoa et al., 1989; Scuka and Mozrzymas, 1992). At the vertebrate neuromuscular junction, desensitization is slow and may not play a significant role in shaping the endplate current or in synaptic depression. However, currents generated by other synaptic receptors often decline rapidly and desensitization is likely to be an important determinant of the amplitude, time-course, and stability of these responses. It is therefore of some physiological importance to understand the molecular events that constitute the desensitization of AChR and other synaptic receptor channels. Katz and Thesleff (1957) observed that at the frog neuromuscular junction, the steady application of ACh virtually abolished the endplate response within seconds, but upon the removal of the agonist, sensitivity recovered rapidly. Because there was no detectable depolarization during recovery, they proposed a cyclic model for AChR activation and desensitization: agonists bind, AChR open and then “desensitize,” and upon washout agonists dissociate and AChR return to their resting condition without reopening. A cyclic reaction scheme implies that unliganded AChR can desensitize. The affinity of desensitized AChR for agonists (Weber et al., 1975; Boyd and Cohen, 1980; Sine and Taylor, 1982) is z 10,000-fold higher than that of resting AChR (Akk and Auerbach, 1996; Wang et al., 1997), but may be similar to that of open AChR (Colquhoun and Sakmann, 1985). It is important to note that “desensitization” describes a host of inactivation phenomena that may arise from a spectrum of molecular and cellular processes. There are multiple components to the desensitization time course (Heidmann and Changeux, 1979). The main component that was first studied in detail by Katz and Thesleff (1957) occurs on a time scale of seconds, but faster (milliseconds; Sakmann et al., 1980; Magleby and Palotta, 1981; Dilger and Brett, 1990) and slower (minutes; Feltz and Trautman, 1982; Chestnut, 1993) components have been identified. Here, we focus on the component that occurs on the 0.1–1-s time scale. Several electrophysiological studies have been done regarding the kinetics of this component of AChR desensitization and recovery. Cachelin and Colquhoun (1989; frog muscle) confirmed the cyclic reaction mechanism and speculated that desensitization occurs exclusively from the diliganded, open conformation. They proposed that the rate limiting step to recovery upon washout is the agonist-independent isomerization of the receptor. Dilger and Liu (1992; mouse BC3H1 cells) found that desensitization closely paralleled the open probability of the channel and used the cyclic Address correspondence to Anthony Auerbach, Department of Physiology and Biophysics, 120 Cary Hall, SUNY, Buffalo, NY 14214. Fax: 716-829-2415; E-mail: [email protected] 1 Abbreviation used in this paper: AChR, acetylcholine receptors. on Jne 0, 2017 D ow nladed fom Published August 1, 1998