Saturation and Microsecond Gating of Current Indicate Depletion-induced Instability of the MaxiK Selectivity Filter

Abstract

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed with a high temporal resolution (50-kHz filter) in symmetrical solutions with 50, 150, or 400 mM KCl and 2.5 mM CaCl2 and 2.5 mM MgCl2. At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca2+ or Mg2+. The analysis of the amplitude histograms by beta distributions revealed that current in this voltage range was reduced by two effects: rate limitation at the cytosolic side of the pore and gating with rate constants 10–20-fold higher than the cutoff frequency of the filter (i.e., dwell times in the microsecond range). The data were analyzed in terms of a model that postulates a coupling between both effects; if the voltage over the selectivity filter withdraws ions from the cavity at a higher rate than that of refilling from the cytosol, the selectivity filter becomes instable because of ion depletion, and current is interrupted by the resulting flickering. The fit of the IV curves revealed a characteristic voltage of 35 mV. In contrast, the voltage dependence of the gating factor R, i.e., the ratio between true and apparent single-channel current, could be fitted by exponentials with a characteristic voltage of 60 mV, suggesting that only part of the transmembrane potential is felt by the flux through the selectivity filter.

Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel

“…However, from a distance, CTX and imipramine produce the same end effect postulated by the footin-the-door mechanism: prolonged occupancy of the ion binding sites at the filter gate. In addition, the proposed lid mechanism is complementary to the "ion depletion of the pore" hypothesis previously postulated to give rise to flickering and instability of the conductive conformation of the filter gate of other K ϩ channels (24, 41,65,66). As presented, the model implies that the filter gate transits between conductive and nonconductive conformations and that the conformation of the gate depends stringently on the occupancy of the filter by either water or a permeant ion (24).…”

How to resolve microsecond current fluctuations in single ion channels: The power of beta distributions

“…9D). 32 The exponential increase of the gating factor I true /I app D (k OC Ck CO )/k CO and especially its dependence on K C concentration (Fig. 9D) has led to a hypothesis, which allows to relate function to structure.…”

“…47 The SNR of optimized single-channel patch clamp experiments allows a maximum bandwidth of around 30-50 kHz. 32,48 In bilayer experiments, the larger membrane area, and thus, the higher membrane capacitance, cause more noise, further limiting the reasonable bandwidth. The lower access resistance in the open bilayer chamber as compared to a narrow patch electrode partly relieves this problem.…”

“…2A and thus has to be determined by the analysis. 32,78 The viral K C channel Kcv NTS shows flickering at positive and negative membrane voltages. 79 At negative voltages, the resulting open-channel noise is extreme (arrow "g" in Fig.…”

“…2) are more the realm of biophysicists: whereas these fast processes may at first glance not be immediately relevant for physiological function, their analysis provides crucial clues to the understanding of stability and dynamics of ion channels and their interaction with blockers or other molecules. 24,25,32,33 Furthermore, computational approaches like molecular dynamics simulations are becoming increasingly powerful, and it is expected that they will be able to predict gating dynamics ( Fig. 1) based on the dynamics of individual chemical interactions or conformational changes in a protein.…”