Science Topics – 184
The function of proteins, including ion channels, is governed by conformational dynamics that span a vast range of timescales. Although single-channel recording is a powerful technique for observing discrete transitions between open and closed states (Figure, upper trace), its temporal resolution is finite. Extremely rapid gating transitions that exceed this limit are not resolved as individual events, but instead manifest as “flicker,” a noise-like current fluctuation (Fig., lower trace). Conventional time-domain analysis methods cannot extract kinetic information from such flickers, leaving the critical aspect of channel dynamics inaccessible.
To overcome this limitation, we developed the BPoF method (Beta-distribution-based analysis for adaptive Post-filtering), which represents a paradigm shift from time-domain to amplitude-domain analysis2. In this approach, the recorded current trace is processed with a digital low-pass filter using a cutoff frequency far below the recording bandwidth. While this step heavily dampens the trace, seemingly destroying information (Fig., colored traces), it is precisely this operation that converts the kinetic information buried in the time domain into the shape of the amplitude histogram of the filtered current. Remarkably, as the filter's cutoff frequency decreases, the shape of the histogram systematically evolves, a transformation that is perfectly described by the beta distribution (Fig. 3D plot). By fitting these histograms, we can accurately determine the rate constants of the underlying flicker gating, even for kinetics that far exceed the recording bandwidth.
Furthermore, we extended this approach to the Multi-fc BPoF method, which analyzes a series of histograms generated at multiple cutoff frequencies. This multidimensional analysis led to a striking discovery: seemingly chaotic flicker can arise from channel switching between two distinct fast-flicker modes with different kinetic properties1. Our method successfully deconvolves the kinetics of both flicker modes and the transitions between them, providing unprecedented insight into the modal gating of the channel. This analytical framework, distinct from conventional time-domain approaches, such as Hidden Markov Models, provides a powerful new tool for exploring the complex conformational dynamics that govern ion channel function and those altered in channelopathies, offering a new window into the molecular basis of physiological regulation and disease.
1. Oiki, S.: Single-Channel Fast-Flicker Currents Deciphered for Kinetic Model Topology and Rates. Cell Reports Physical Science 6, 102842, 2025
2. Yoshida, T, Oiki, S.: Resolving Protein’s Conformational Kinetics from Single-Molecule Fast Flicker Data. Cell Reports Physical Science 5, 101925, 2024.
＜Figure Legends＞
Analysis of single-channel flicker currents using BPoF method. The upper trace shows slow gating with discrete transitions that are amenable to the conventional time-domain analysis. The lower trace shows fast flicker gating, which is the target of the proposed method. When a digital low-pass filter (lower left) is applied to the recorded current, the resulting traces are heavily dampened (violet, green, and orange). However, the corresponding amplitude histograms systematically evolve from bimodal (violet) to skewed (green) and finally to a unimodal shape (red) as the cut-off frequency is lowered. This family of shapes is perfectly described by the theoretical beta distribution (3D plot), and fitting these histograms allows for the determination of fast gating rate constants. Furthermore, the Multi-fc BPoF method can decipher hidden transitions between two distinct kinetic modes (mode switching, schematic lower right) within the flicker.
Biomedical Imaging Research Center, University of Fukui, Japan