Thompson J and Begenisich T (2005), Two stable, conducting conformations of the sel...

XB-ART-1888

J Gen Physiol 2005 Jun 01;1256:619-29. doi: 10.1085/jgp.200509251.

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Two stable, conducting conformations of the selectivity filter in Shaker K+ channels.

Thompson J , Begenisich T .

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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.

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	Figure 1. . Low internal K+ reveals two components of external TEA block. Fraction of Shaker K+ channel current blocked by external TEA in 50 mM (A) and 5 mM (B) internal K+ at â10 mV (top) and +70 mV (bottom). Data points are the mean values from 3 to 6 measurements with standard error limits for those whose errors are larger than the symbol size. Insets show raw currents in the absence (Control) and presence of 50 mM external TEA. Calibrations: (A) â10 mV, 50 pA/10 ms; +70 mV, 0.2 nA/10 ms; (B) â10 mV, 50 pA/10 ms; +70 mV, 0.1 nA/10 ms. Dashed lines are fits of Eq. 1 to the data with Kapp values of (A) â10 mV, 20 mM; +70 mV, 43 mM; (B) â10 mV, 32 mM; +70 mV, 89 mM. Solid lines are fits of Eq. 2 to the data with the A, Kapplow, and Kapphigh parameter values shown in Fig. 2.
	Figure 2. . Voltage dependence of the two components of external TEA block. (A) The fraction of current block by external TEA with high affinity (the parameter A in Eq. 2) at 50 mM (â) and 5 mM (âª) internal K+. (B) The Kapphigh value from Eq. 2 determined in 50 mM (â) and 5 mM (âª) internal K+. (C) The Kapplow value from Eq. 2 determined in 50 mM (â) and 5 mM (âª) internal K+. Insets: Kapphigh and Kapplow values obtained at negative potentials from block of current tails with an activation voltage of +20 mV (see materials and methods). The values at +20 mV were from block of steady-state current during the activation pulse.
	Figure 3. . Properties of the two components of external TEA block. (A) Fraction of high-affinity block as a function of internal K+ with 5 mM external K+ (âª) except for the 2 mM data, which were obtained with 2 mM external K+. Data were also obtained with 20 mM external and 20 mM internal K+ (). The solid line is a fitted spline function and serves only to connect the data points. (B) Voltage dependence of the Kapphigh values at the indicated internal K+ concentrations (different symbol for each internal K+ level) with 5 mM external K+ except for the 2 mM data, which were obtained with 2 mM external K+. Data were also obtained with 20 mM external and 20 mM internal K+ (). The solid lines are fits of Eq. 3a to the data all with a KT2 value of 5 mM and the Î´2 and Keq2(0) values shown in Fig. 5. (C) Voltage dependence of the Kapplow values at the indicated internal K+ levels with 5 mM external K+ (same symbols as in B) except for the 2 mM data, which were obtained with 2 mM external K+. Data were also obtained with 20 mM external and 20 mM internal K+ (*). Solid line is from Eq. 3b with KT1, Î´1, and Keq1(0) values of 70 mM, 0.78, and 0.47, respectively. The dashed line associated with the 5 mM K+ data (â¿) is from Eq. 3b with KT1, Î´1, and Keq1(0) values of 70 mM, 0.5, and 2, respectively. The dashed line associated with the 2 mM K+ data (â¦) is from Eq. 3b with KT1, Î´1, and Keq1(0) values of 70 mM, 0.52, and 2.1, respectively.
	Figure .
	Figure .
	Figure 4. . Determination of the affinity and apparent voltage dependence for K+ binding to an internal site. (A) Fraction of current at â10 mV (top) and +70 mV (bottom) blocked by the indicated internal TEA concentrations in the absence (â¢) and presence (â) of 100 mM external TEA with 135 mM internal K+. Symbols with standard error limits represent mean values from 3â4 measurements. Symbols without error bars represent individual measurements. Solid lines are fits of Eq. 1 to the data with Kapp values at â10 mV of 0.65 and 1.4 mM and at +70 mV of 0.39 and 1.2 mM in the absence and presence of external TEA, respectively. (B) The ratio of the Kapp values for internal TEA block in the presence and absence of external TEA with 135 mM internal K+ (âª) and with the internal K+ replaced by Rb+ (â). The solid line with the internal K+ data is the best fit of Eqs. 3 and 4 to the data with KK(0) and Î´ values of 102 mM and 0.17, respectively. The line connecting the Rb+ data has no theoretical meaning.
	Figure 5. . Properties of selectivity filter one-ion and two-ion occupancy modes. Electrical distance and equilibrium values from fits of Eqs. 3a or 3b to the relevant data in Fig. 3. (A) Internal K+ and the electrical distance for one-ion (Î´1, â¢) and two-ion (Î´2, â) occupancy modes. Dashed lines represent the mean of the electrical distance parameters. (B) Internal K+ and the equilibrium values (at 0 mV) for the one-ion (K eq1(0), â¢) and two-ion (K eq2(0), â) occupancy modes for internal K+ levels â¥20 mM. * represents K eq2(0) at 10 mM internal K+. The dashed line represents the mean of the K eq1(0) values.

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