Single-channel fast-flicker currents deciphered for kinetic model topology and rates

Single-molecule measurements of protein dynamics reveal discrete transitions between conformational states, providing critical kinetic information. However, recording signals often elicit flickering because rapid conformational transitions exceed the temporal recording resolution, making time-domain kinetic analysis challenging. We developed an amplitude-domain method to decipher the underlying rate of channel flickering. Experimental single-channel currents, when passed through a first-order filter, often yield two beta distributions (double-beta distributions) in the amplitude histogram. We revealed that these two components were projected from current traces comprising two aggregated Markov processes emerging alternatively (double-flicker gating). The underlying gating model of double flickering is related to the model topology, which exhibits mode switching. To estimate the underlying double-flickering rates, multiple amplitude histograms drawn from the filtered current data at different cut-off frequencies were simultaneously fitted with double-beta distributions. The simulated data for various models and rates verified the capability of the method for robust rate estimation.