Improved sub-µs gating analysis indicates conformational changes as caused by ion/pore interactions in the MaxiK channel

This thesis aims at the analysis of fast gating events in ion channels. Experiments were done on single MaxiK channels expressed in HEK cells using the patch clamp technique in voltage clamp mode. Crucial for high temporal resolution was the signal-to-noise ratio which was improved by optimizing the set-up and pipette treatment. Furthermore, temporal resolution was increased by sharpening the mathematical tools employed in the analysis of the kinetic behavior of single-channel currents. This was done along four lines. 1. Splitting amplitude histograms into distributions-per-level and investigating their properties with respect to model discrimination, determination of the rate constants of fast gating and the detection of failures of jump detection. 2. Creating the SQ fit, a two-step fit algorithm consisting of a direct fit of patch clamp time series, by means of a 1-step prediction algorithm and a fit of the distribution-per-open-level by means of beta distributions. 3. Finding and testing the ability of the beta fit to determine the true single-channel current in time series where the apparent current was attenuated by averaging over undetected fast gating. 4. Comparing the SQ fit with the 2-dimensional dwell-time fit as improved by T. Huth. After having developed the new tools, they were applied to the analysis of fast gating events. Two phenomena could gain the outmost benefit from the new analytical tools: 1. The negative slope in the current-voltage curve occurring at positive potentials in symmetrical solutions with K+ as the only monovalent cation. 2. The negative slope occurring at negative potentials in luminal K+/Tl+ solution when the current dragged Tl+ into the channel. Both kinds of measurement resulted in insights about ion/protein interaction. The key for the creation of molecular models of ion/channel interactions was found in the experiments with K+ as the sole monovalent cation. In the range of the negative slope, saturation of the true single-channel current and onset of fast gating occurred in the same voltage range. This finding suggested a mechanistic model: On the cytosolic side, diffusion limitation produced saturation of outward-current. This caused ion depletion in the selectivity filter. The idea that gating occurred when permeant ions did no longer compensate the repulsive forces of the carbonyl groups was supported by MD simulations reported in the literature. The model correctly predicted the effect of different K+ concentrations. For explaining the negative slope induced by negative membrane potentials when luminal Tl+ was dragged into the channel, the basic model had to be extended. Here, the effect of luminal diffusion limitation on voltage-induced gating was not sufficient for explaining the experimental findings. This effect had to be modulated by additional Tl+ binding sites in the cavity or at the inner side of the selectivity filter. Describing the full set of gating phenomena induced by Tl+ would require the analysis of the “Great Markov Model” of the MaxiK channel. It is shown by preliminary approaches with classical tools what kind of insights would be obtained from this kind of analysis and what kind of problems keep the ratio “scientific output/time” at a very low level. Nevertheless, examples are given that the analysis of beta distributions can be helpful for the extension of the Markov model. Especially the picture of a rattly channel has arisen, implying that open states are not really open and closed states are not really closed. Finally, it is discussed that using the viral channel Kcv instead of MaxiK offers better chances to continue the building of a bridge between electrophysiology and structural biology as has been initiated in this thesis.