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

Thompson J, Begenisich T - J. Gen. Physiol. (2005)

Bottom Line: 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.This distribution does not change with internal K(+).Our analysis further suggests that the four K(+) sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, NY 14642, USA.

ABSTRACT
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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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 (δ1, •) and two-ion (δ2, ○) 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), •) and two-ion (K eq2(0), ○) occupancy modes for internal K+ levels ≥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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fig5: 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 (δ1, •) and two-ion (δ2, ○) 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), •) and two-ion (K eq2(0), ○) occupancy modes for internal K+ levels ≥20 mM. * represents K eq2(0) at 10 mM internal K+. The dashed line represents the mean of the K eq1(0) values.

Mentions: 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+ (▪) 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 δ2 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, δ1, and Keq1(0) values of 70 mM, 0.78, and 0.47, respectively. The dashed line associated with the 5 mM K+ data (▿) is from Eq. 3b with KT1, δ1, and Keq1(0) values of 70 mM, 0.5, and 2, respectively. The dashed line associated with the 2 mM K+ data (♦) is from Eq. 3b with KT1, δ1, and Keq1(0) values of 70 mM, 0.52, and 2.1, respectively.


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

Thompson J, Begenisich T - J. Gen. Physiol. (2005)

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 (δ1, •) and two-ion (δ2, ○) 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), •) and two-ion (K eq2(0), ○) occupancy modes for internal K+ levels ≥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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getmorefigures.php?uid=PMC2234082&req=5

fig5: 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 (δ1, •) and two-ion (δ2, ○) 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), •) and two-ion (K eq2(0), ○) occupancy modes for internal K+ levels ≥20 mM. * represents K eq2(0) at 10 mM internal K+. The dashed line represents the mean of the K eq1(0) values.
Mentions: 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+ (▪) 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 δ2 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, δ1, and Keq1(0) values of 70 mM, 0.78, and 0.47, respectively. The dashed line associated with the 5 mM K+ data (▿) is from Eq. 3b with KT1, δ1, and Keq1(0) values of 70 mM, 0.5, and 2, respectively. The dashed line associated with the 2 mM K+ data (♦) is from Eq. 3b with KT1, δ1, and Keq1(0) values of 70 mM, 0.52, and 2.1, respectively.

Bottom Line: 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.This distribution does not change with internal K(+).Our analysis further suggests that the four K(+) sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, NY 14642, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus