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Correspondence to Ted Begenisich: ted_begenisich{at}URMC.rochester.edu
We have examined the voltage dependence of external TEA block of Shaker K+ channels over a range of internal K+ concentrations from 2 to 135 mM. We found that the concentration dependence of external TEA block in low internal K+ solutions could not be described by a single TEA binding affinity. The deviation from a single TEA binding isotherm was increased at more depolarized membrane voltages. The data were well described by a two-component binding scheme representing two, relatively stable populations of conducting channels that differ in their affinity for external TEA. The relative proportion of these two populations was not much affected by membrane voltage but did depend on the internal K+ concentration. Low internal K+ promoted an increase in the fraction of channels with a low TEA affinity. The voltage dependence of the apparent high-affinity TEA binding constant depended on the internal K+ concentration, becoming almost voltage independent in 5 mM. The K+ sensitivity of these low- and high-affinity TEA states suggests that they may represent one- and two-ion occupancy states of the selectivity filter, consistent with recent crystallographic results from the bacterial KcsA K+ channel. We therefore analyzed these data in terms of such a model and found a large (almost 14-fold) difference between the intrinsic TEA affinity of the one-ion and two-ion modes. According to this analysis, the single ion in the one-ion mode (at 0 mV) prefers the inner end of the selectivity filter twofold more than the outer end. This distribution does not change with internal K+. The two ions in the two-ion mode prefer to occupy the inner end of the selectivity filter at low K+, but high internal K+ promotes increased occupancy of the outer sites. Our analysis further suggests that the four K+ sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.
Key Words: Shaker • K channels • TEA • ion occupancy • selectivity filter
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