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Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+).

Horrigan FT, Cui J, Aldrich RW - J. Gen. Physiol. (1999)

Bottom Line: However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e).These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors.These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.

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Steady state activation. (A) A family of IK evoked in response to 20-ms depolarizations (+80 to +280 in 20-mV steps, holding potential = −80, 20°C). GK(V) was determined by measuring the tail current amplitude at −80 mV immediately after each pulse. (B) The GK–V relationships from 23 experiments (○) were normalized by GKmax and shifted along the voltage axis to align half-activation voltages (see methods). The average G–V (•) represents the mean ± SEM of the normalized-shifted data determined over 15-mV intervals. A Boltzmann function raised to a power of 3.2 (z = 0.69 e, solid line) represents the best fit to the individual data, excluding experiments where Vmax < 240 mV. GKmax could not be directly determined for the excluded experiments; therefore, these data were normalized based on the Boltzmann3.2 fit. Dashed lines indicate predictions of Fig. 5 [∈ = δ/γ, ∈(0) = 0.367, z∈ = 0.295 e, zJ = 0.55 e, Vh(J) = 145] and Fig. 9 [L(0) = 2 e−6, zL = 0.4 e, zJ = 0.55 e, Vh(J) = 145, D = 17]. (C) The data are replotted on a semi-log scale together with the Boltzmann3.2 fit (solid line). The maximum voltage dependence of G–V is indicated by a dashed line (z = 2.0 e). (D) Single channel currents were recorded at the indicated voltages and filtered at 20 kHz. The corresponding all-points histograms are plotted on a semi-log scale (points versus picoamperes). (E) Normalized open probability (Po/Pomax, see text) determined from single channel currents is plotted versus voltage for several experiments in 0 Ca2+ (filled symbols) or 4.5 μM Ca2+ (open symbols). The dashed line indicates the maximum voltage dependence of the macroscopic G–V from C. (F) The normalized Po–V relationship from −120 to +300 mV combines the macroscopic and single channel data. Filled symbols indicate averages (mean ± SEM, 15-mV bin width), while open symbols represent data from individual experiments. Predictions of Fig. 5 (dashed line) and IX (solid line) are the same as in B.
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Figure 6: Steady state activation. (A) A family of IK evoked in response to 20-ms depolarizations (+80 to +280 in 20-mV steps, holding potential = −80, 20°C). GK(V) was determined by measuring the tail current amplitude at −80 mV immediately after each pulse. (B) The GK–V relationships from 23 experiments (○) were normalized by GKmax and shifted along the voltage axis to align half-activation voltages (see methods). The average G–V (•) represents the mean ± SEM of the normalized-shifted data determined over 15-mV intervals. A Boltzmann function raised to a power of 3.2 (z = 0.69 e, solid line) represents the best fit to the individual data, excluding experiments where Vmax < 240 mV. GKmax could not be directly determined for the excluded experiments; therefore, these data were normalized based on the Boltzmann3.2 fit. Dashed lines indicate predictions of Fig. 5 [∈ = δ/γ, ∈(0) = 0.367, z∈ = 0.295 e, zJ = 0.55 e, Vh(J) = 145] and Fig. 9 [L(0) = 2 e−6, zL = 0.4 e, zJ = 0.55 e, Vh(J) = 145, D = 17]. (C) The data are replotted on a semi-log scale together with the Boltzmann3.2 fit (solid line). The maximum voltage dependence of G–V is indicated by a dashed line (z = 2.0 e). (D) Single channel currents were recorded at the indicated voltages and filtered at 20 kHz. The corresponding all-points histograms are plotted on a semi-log scale (points versus picoamperes). (E) Normalized open probability (Po/Pomax, see text) determined from single channel currents is plotted versus voltage for several experiments in 0 Ca2+ (filled symbols) or 4.5 μM Ca2+ (open symbols). The dashed line indicates the maximum voltage dependence of the macroscopic G–V from C. (F) The normalized Po–V relationship from −120 to +300 mV combines the macroscopic and single channel data. Filled symbols indicate averages (mean ± SEM, 15-mV bin width), while open symbols represent data from individual experiments. Predictions of Fig. 5 (dashed line) and IX (solid line) are the same as in B.

Mentions: In all cases Pk was well fit by a Poisson distribution and the values of nPo obtained by the two methods differed by <5%. This is consistent with the idea that IK represents the activity of a large population of channels with low Po rather than a subpopulation with higher Po. Normalized open probability (Po/Pomax = nPo/nPomax) was determined by combining nPo measurements with an estimate of nPomax obtained from the macroscopic GK–V relationship in the same patch (nPomax = GKmax/gK, where gK is the single channel conductance). Patches that were used to measure single channel activity at negative voltages often produced currents that were too large to measure (>20 nA) at voltages that activate mSlo channels maximally. In these cases, Gmax was estimated by fitting the macroscopic GK-V with a Boltzmann function ({1 + exp[−ze(V − Vh)/kT]}−1) raised to the 3.2 power as in Fig. 6B and Fig. C.


Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+).

Horrigan FT, Cui J, Aldrich RW - J. Gen. Physiol. (1999)

Steady state activation. (A) A family of IK evoked in response to 20-ms depolarizations (+80 to +280 in 20-mV steps, holding potential = −80, 20°C). GK(V) was determined by measuring the tail current amplitude at −80 mV immediately after each pulse. (B) The GK–V relationships from 23 experiments (○) were normalized by GKmax and shifted along the voltage axis to align half-activation voltages (see methods). The average G–V (•) represents the mean ± SEM of the normalized-shifted data determined over 15-mV intervals. A Boltzmann function raised to a power of 3.2 (z = 0.69 e, solid line) represents the best fit to the individual data, excluding experiments where Vmax < 240 mV. GKmax could not be directly determined for the excluded experiments; therefore, these data were normalized based on the Boltzmann3.2 fit. Dashed lines indicate predictions of Fig. 5 [∈ = δ/γ, ∈(0) = 0.367, z∈ = 0.295 e, zJ = 0.55 e, Vh(J) = 145] and Fig. 9 [L(0) = 2 e−6, zL = 0.4 e, zJ = 0.55 e, Vh(J) = 145, D = 17]. (C) The data are replotted on a semi-log scale together with the Boltzmann3.2 fit (solid line). The maximum voltage dependence of G–V is indicated by a dashed line (z = 2.0 e). (D) Single channel currents were recorded at the indicated voltages and filtered at 20 kHz. The corresponding all-points histograms are plotted on a semi-log scale (points versus picoamperes). (E) Normalized open probability (Po/Pomax, see text) determined from single channel currents is plotted versus voltage for several experiments in 0 Ca2+ (filled symbols) or 4.5 μM Ca2+ (open symbols). The dashed line indicates the maximum voltage dependence of the macroscopic G–V from C. (F) The normalized Po–V relationship from −120 to +300 mV combines the macroscopic and single channel data. Filled symbols indicate averages (mean ± SEM, 15-mV bin width), while open symbols represent data from individual experiments. Predictions of Fig. 5 (dashed line) and IX (solid line) are the same as in B.
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Figure 6: Steady state activation. (A) A family of IK evoked in response to 20-ms depolarizations (+80 to +280 in 20-mV steps, holding potential = −80, 20°C). GK(V) was determined by measuring the tail current amplitude at −80 mV immediately after each pulse. (B) The GK–V relationships from 23 experiments (○) were normalized by GKmax and shifted along the voltage axis to align half-activation voltages (see methods). The average G–V (•) represents the mean ± SEM of the normalized-shifted data determined over 15-mV intervals. A Boltzmann function raised to a power of 3.2 (z = 0.69 e, solid line) represents the best fit to the individual data, excluding experiments where Vmax < 240 mV. GKmax could not be directly determined for the excluded experiments; therefore, these data were normalized based on the Boltzmann3.2 fit. Dashed lines indicate predictions of Fig. 5 [∈ = δ/γ, ∈(0) = 0.367, z∈ = 0.295 e, zJ = 0.55 e, Vh(J) = 145] and Fig. 9 [L(0) = 2 e−6, zL = 0.4 e, zJ = 0.55 e, Vh(J) = 145, D = 17]. (C) The data are replotted on a semi-log scale together with the Boltzmann3.2 fit (solid line). The maximum voltage dependence of G–V is indicated by a dashed line (z = 2.0 e). (D) Single channel currents were recorded at the indicated voltages and filtered at 20 kHz. The corresponding all-points histograms are plotted on a semi-log scale (points versus picoamperes). (E) Normalized open probability (Po/Pomax, see text) determined from single channel currents is plotted versus voltage for several experiments in 0 Ca2+ (filled symbols) or 4.5 μM Ca2+ (open symbols). The dashed line indicates the maximum voltage dependence of the macroscopic G–V from C. (F) The normalized Po–V relationship from −120 to +300 mV combines the macroscopic and single channel data. Filled symbols indicate averages (mean ± SEM, 15-mV bin width), while open symbols represent data from individual experiments. Predictions of Fig. 5 (dashed line) and IX (solid line) are the same as in B.
Mentions: In all cases Pk was well fit by a Poisson distribution and the values of nPo obtained by the two methods differed by <5%. This is consistent with the idea that IK represents the activity of a large population of channels with low Po rather than a subpopulation with higher Po. Normalized open probability (Po/Pomax = nPo/nPomax) was determined by combining nPo measurements with an estimate of nPomax obtained from the macroscopic GK–V relationship in the same patch (nPomax = GKmax/gK, where gK is the single channel conductance). Patches that were used to measure single channel activity at negative voltages often produced currents that were too large to measure (>20 nA) at voltages that activate mSlo channels maximally. In these cases, Gmax was estimated by fitting the macroscopic GK-V with a Boltzmann function ({1 + exp[−ze(V − Vh)/kT]}−1) raised to the 3.2 power as in Fig. 6B and Fig. C.

Bottom Line: However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e).These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors.These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.

Show MeSH
Related in: MedlinePlus