Desensitization of diliganded mouse muscle nicotinic acetylcholine receptor channels

Nicotinic ACh receptor channels (AChRs) exposed to high concentrations of ACh adopt 'desensitized' conformations that have a high affinity for the transmitter and no measurable ion conductance. Single-channel currents elicited by 0.1 or 1 mM ACh were recorded from human embryonic kidney (HEK) cells that had been transiently transfected with mouse alpha, beta, delta, and epsilon subunits. On the time scale of approximately 0.1 ms to approximately 1 h, apparent open intervals are described by a single exponential component, and shut intervals associated with desensitization are described by the sum of four or five exponential components. The kinetic behaviour appeared to be stationary and homogeneous. Desensitization rate constants were estimated by kinetic modelling of currents from cell-attached and outside-out patches (where the number of channels in the patch was measured). A single AChR recovered from the longest-lived desensitized state only after approximately 5 min. The occupancy of an AChR for each of the desensitized states was calculated as a function of time after the continuous application of a pulse of saturating ACh. The longest-lived desensitized state accounted for 90 % of the total only after several seconds. The fractional recovery from desensitization (during a 200 ms wash period) decreased as the duration of the desensitizing pulse increased, suggesting that recovery is slower from the longer-lived desensitized states. The free energy landscape for the AChR desensitization reaction in cell-attached patches exhibited an initial destabilization, followed by a plateau region of gradually increasing stability, followed by a deep well.