Two-dimensional probability density analysis of single channel currents from reconstituted acetylcholine receptors...

Two-dimensional probability density analysis of single channel current recordings was applied to two purified channel proteins reconstituted in planar lipid bilayers: Torpedo acetylcholine receptors and voltage-sensitive sodium channels from rat brain. The information contained in the dynamic history of the gating process, i.e., the time sequence of opening and closing events was extracted from two-dimensional distributions of transitions between identifiable states. This approach allows one to identify kinetic models consistent with the observables. Gating of acetylcholine receptors expresses "memory" of the transition history: the receptor has two channel open (O) states; the residence time in each of them strongly depends on both the preceding open time and the intervening closed interval. Correspondingly, the residence time in the closed (C) states depends on both the preceding open time and the preceding closed time. This result confirms the scheme that considers, at least, two transition pathways between the open and closed states and extends the details of the model in that it defines that the short-lived open state is primarily entered from long-lived closed states while the long-lived open state is accessed mainly through short-lived closed states. Since ligand binding to the acetylcholine-binding sites is a reaction with channel closed states, we infer that the longest closed state (approximately 19 ms) is unliganded, the intermediate closed state (approximately 2 ms) is singly liganded and makes transitions to the short open state (approximately 0.5 ms) and the shortest closed state (approximately 0.4 ms) is doubly liganded and isomerizes to long open states (approximately 5 ms). This is the simplest interpretation consistent with available data. In contrast, sodium channels modified with batrachotoxin to eliminate inactivation show no correlation in the sequence of channel opening and closing events, i.e., have no memory of the transition history. This result is, therefore, consistent with any kinetic scheme that considers a single transition pathway between open and closed states, and confirms the C-C-O model previously inferred from one-dimensional distribution analysis. The strategy described should be of general validity in the analysis of single channel events from channel proteins in both natural and reconstituted membranes.