[51] Stationary single-channel analysis

doi:10.1016/0076-6879(92)07053-Q

Methods in Enzymology

Volume 207, 1992, Pages 729-746

https://doi.org/10.1016/0076-6879(92)07053-Q Get rights and content

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Analysis of records of single-channel current can provide insight into the mechanisms by which channels open and close. This chapter describes several useful methods of kinetic analysis of stationary single-channel data. In a stationary system, the properties of the ensemble do not change with time; the mean probabilities of occupancy of specific configurations remain constant. Single-channel kinetic analysis can be divided into three distinct steps. The first step involves the theoretical derivation of predictions of kinetic models. The second step of single-channel kinetic analysis is to extract something from real data that can be compared to theoretical predictions. These operations generally start with raw single-channel data and generate open time and closed time histograms, or more complicated distillates of channel behavior. One thus obtains an experimental result in a form that can be directly compared with a theoretical prediction. This comparison, which most often involves some form of curve fit, is the third and final step of single-channel kinetic analysis.

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Cited by (18)

Full-fusion and kiss-and-run in chromaffin cells controlled by irreversible vesicle size-dependent fusion pore transitions
2022, Cell Calcium
Citation Excerpt :
Irreversibility implies a violation of detailed balance, which is a requirement for negative exponential coefficients in lifetime distributions (Fig. 3) [31]. These ideas draw on an extensive body of literature about the stochastic nature of transitions of ion channels [32,35,46]. However, ion channel gating is reversible in the vast majority of cases, and single-channel lifetime distributions nearly always have all-positive exponential coefficients.
Exocytosis operates through two distinct modes. Full-fusion leads to rapid expulsion of the entire content of a vesicle; kiss-and-run leads to slow and partial expulsion. These two modes have important biological consequences for endocrine regulation and synaptic transmission. Amperometry recordings of catecholamine release from chromaffin cells reveal single-vesicle fusion events corresponding to both of these modes, but classification is often difficult. This study introduces a new method of analyzing amperometry data to improve this classification. The ratio of the average amplitude to the peak amplitude differs between full-fusion and kiss-and-run, and the probability distribution of this ratio is well fitted by a double-Gaussian. Kiss-and-run events identified by this method have fusion pores with kinetic properties different from pores associated with full-fusion. They have slower transition rates and lifetime distributions indicative of irreversible transitions. The total-charge of an amperometric spike is expected to scale with vesicle volume during a full-fusion event. The cube root of this quantity should therefore scale with diameter, but the distribution of this quantity differs from the distribution of vesicle diameter seen in the electron microscope. Fusion pore lifetimes associated with full-fusion depend on vesicle size, and this makes the choice of mode size dependent. The fusion pore thus bifurcates after opening, and vesicle size influences this choice. The secretory vesicle protein synaptophysin influences the size dependence of fusion pore lifetime and the choice of release mode. Incorporating vesicle size into an analysis of release mode reconciled the kinetics of fusion pores, as well as the distributions of vesicle diameter and catecholamine content. Thus, the initial fusion pore emerges as a critical focus in endocrine regulation. By modulating the size-dependence of the mode of exocytosis, changes in the molecular makeup of the exocytotic apparatus can impact the shape and size of an amperometric event, and the speed and composition of secretion.
Markov chain inversion approach to identify the transition rates of ion channels
2012, Acta Mathematica Scientia
We consider how to identify the transition rates of ion channels with the underlying scheme which is kinetically modelled as time-homogeneous Markov chain. A Markov chain inversion approach is developed to perform a difficult inversion to identify the transition rates from the parameters characterizing the lifetime distributions at a small number of states, although it is straightforward to derive the lifetime distribution. The general explicit equations relating the parameters of the lifetime distribution to the transition rates are derived and transition rates are then obtained as roots to this system of equations. The concrete solutions are proposed to the basic and regular schemes such as linear, star-graph branch and loop. Useful conclusions and solutions to realistic schemes are also included to show its efficiency.
In search of the fusion pore of exocytosis
2007, Biophysical Chemistry
Research on calcium-triggered exocytosis has converged on the fusion pore as a critical kinetic intermediate. Using sensitive biophysical methods to record signals from living cells in the act of releasing neurotransmitter or hormone has provided clues about the structure and composition of fusion pores. The dynamics of fusion pore opening, closing, and dilating has revealed how specific proteins transduce a calcium binding signal to catalyze membrane fusion. The fusion pore determines how rapidly neurotransmitter is expelled from a vesicle into the synaptic cleft. This rate places constraints on the form of a synaptic response during different modes of release.
Fusion pore dynamics are regulated by synaptotagmin•t-SNARE interactions
2004, Neuron
Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca²⁺] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca²⁺ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca²⁺-triggered syt•SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca²⁺-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.
Inversion of Markov processes to determine rate constants from single- channel data
1997, Biophysical Journal
The determination of rate constants from single-channel data can be very difficult, in part because the single-channel lifetime distributions commonly analyzed by experimenters often have a complicated mathematical relation to the channel gating mechanism. The standard treatment of channel gating as a Markov process leads to the prediction that lifetime distributions are exponential functions. As the number of states of a channel gating scheme increases, the number of exponential terms in the lifetime distribution increases, and the weights and decay constants of the lifetime distributions become progressively more complicated functions of the underlying rate constants. In the present study a mathematical strategy for inverting these functions is introduced in order to determine rate constants from single-channel lifetime distributions. This inversion is easy for channel gating schemes with two or fewer states of a given conductance, so the present study focuses on schemes with more states. The procedure is to derive explicit equations relating the parameters of the lifetime distribution to the rate constants of the scheme. Such equations can be derived using the equality between symmetric functions of eigenvalues of a matrix and sums over principle minors, as well as expressions for the moments, derivatives, and weights of a lifetime distribution. The rate constants are then obtained as roots to this system of equations. For a gating scheme with three sequential closed states and a single gateway state, exact analytical expressions were found for each rate constant in terms of the parameters of the three-exponential closed-time distribution. For several other gating schemes, systems of equations were found that could be solved numerically to obtain the rate constants. Lifetime distributions were shown to specify a unique set of real rate constants in sequential gating schemes with up to five closed or five open states. For kinetic schemes with multiple gating pathways, the analysis of simulated data revealed multiple solutions. These multiple solutions could be distinguished by examining two-dimensional probability density functions. The utility of the methods introduced here are demonstrated by analyzing published data on nicotinic acetylcholine receptors, GABA(A) receptors, and NMDA receptors.
Fusion pore stability and dynamics
2022, Exocytosis: From Molecules to Cells

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[51] Stationary single-channel analysis

Publisher Summary

Trends Pharmacol. Sci.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Proc. R. Soc. London, Ser. B