Kinetic analysis of channel gating. Application to the cholinergic receptor channel and the chloride channel from Torpedo californica

Abstract

Identification of the minimum number of ways in which open and closed states communicate is a crucial step in defining the gating kinetics of multistate channels. We used certain correlation functions to extract information about the pathways connecting the open and closed states of the cation channel of the purified nicotinic acetylcholine receptor and of the chloride channel of Torpedo californica electroplax membranes. Single channel currents were recorded from planar lipid bilayers containing the membrane channel proteins under investigation. The correlation functions are conveniently computed from single channel current records and yield information on E, the minimum number of entry/exit states into the open or closed aggregates. E gives a lower limit on the numbers of transition pathways between open and closed states. For the acetylcholine receptor, the autocorrelation analysis shows that there are at least two entry/exit states through which the open and closed aggregates communicate. The chloride channel fluctuates between three conductance substates, here indentified as C, M, and H for closed, intermediate, and high conductance, respectively. Correlation analysis shows that E is greater than or equal to 2 for the M aggregate, indicating that there are at least two distinct entry/exit states in the M aggregate. In contrast, there is no evidence for the existence of more than one entry/exit state in the C or H aggregates. Thus, these correlation functions provide a simple and general strategy to extract information on channel gating kinetics.

Monoclonal antibodies specific to the beta and gamma subunits of the Torpedo acetylcholine receptor inhibit single-channel activity

“…ACh receptor single-channel currents were activated by 10 PM ACh and recorded at 100 mV. The amplitudes A, and A,, and the time constants TV and TV, of the exponential fit obtained by a x2 minimization algorithm (Fletcher, 197 1;Labarca et al. 1985a.…”

Gating Reaction Mechanisms for NMDA Receptor Channels

“…To help ascertain the connectivity of the states in the model, two-dimensional (2D) dependency plot analysis (Magleby and Song, 1992) and open-open, closed-closed, and open-closed autocorrelation analyses Labarca et al, 1985) were performed on idealized single-channel data without correcting for missed events. The autocorrelation coefficient for two intervals T 1 and T 2 in an idealized single-channel current is as follows:…”

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

“…The data imply that the conformational change undergone by the holotoxin at acidic pH (Boquet et al, 1984;cf. Montecucco & Schiavo, 1994) (Labarca et al, 1985;Montal et al, 1986;Gambale & Montal, 1988;Grove et al, 1992). In NaC1, Po = 0.43; in KCI, Po = 0.77.…”

“…The aqueous subphase was composed of 0.5 M KC1 or NaCI, 1 mM CaCI,, and 5 mM Hepes, pH 7.4, unless otherwise indicated. Bilayer experiments were performed at 24 k 2 "C. Acquisition and analysis of single-channel currents were as described (Labarca et al, 1985;Montal et al, 1986;Gambale & Montal, 1988;Grove et al, 1992). Records were filtered with an 8-pole Bessel filter (Frequency Services, Haverhill, Massachusetts) and digitized at 0.5-ms sampling interval using an Axon TL-I interface (Axon Instruments, Burlingame, California) connected to an Everex Step 386 computer (Everex, Fremont, California).…”