Saturation and microsecond gating of current indicate depletion-induced instability of the MaxiK selectivity filter.
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 CaCl(2) and 2.5 mM MgCl(2). At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca(2+) or Mg(2+). 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.
Schroeder
Indra
I
Department of Structural Biology, University of Kiel, 24098 Kiel, Germany.
Hansen
Ulf-Peter
UP
D002460:Cell Line; D004553:Electric Conductivity; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D013997:Time Factors
C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits