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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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Voltage dependence of IK delay. (A) The initial time course of IK activation measured at +180 mV from the same patch as Fig. 2 shows a decreased delay following a 1-ms prepulse to various voltages (−80, 0, 40, 80, 100, and 120 mV). To accurately represent the time course of channel opening, currents during the prepulse were scaled by a factor IK(+180)/IK(Vpre) representing the ratio of single channel current amplitudes measured at the test and prepulse voltages. (B) A family of IK evoked at different voltages (+120 to +240 in 20-mV steps, holding potential = −80) are fit with exponential functions (dashed lines), demonstrating a voltage-dependent change in delay. (C) Delay duration (Δt) is plotted versus pulse voltage for two experiments. The plots are fit by functions of the form Δt = 1/(a + b) with a=ao*ezaekt,b=bo*ezbekt(za = +0.28 e, zb = −0.28 e ; (▴) ao = 124 s−1, bo = 4,767 s−1; (•) ao = 98 s−1, bo = 3,902 s−1). The delay was not well determined at the lowest voltages; therefore, fits were constrained with the simplifying assumption za = zb. (D) The average Δt–V relationship (mean ± SEM, n = 6), was obtained after first normalizing individual plots (see text) to mean Δt measured from +180 to +195 mV. The data are fit by the above function (solid line, za = +0.28 e; zb = −0.28 e ; ao = 119 s−1, bo = 4,240 s−1) and reproduced by Fig. 5 [dashed line: zα = +0.28 e, α(0) = 244 s−1, zβ = −0.28 e, β(0) = 8,670 s−1, zδ = 0.155 e, δ(0) = 49.4 s−1, zγ = −0.155 e, γ(0) = 134 s−1]. The rate constants in Fig. 5 that describe voltage sensor movement (α, β) are 2.05-fold greater than (a, b) at all voltages. Thus Δt = 1/(a + b) is proportional the voltage-sensor time constant τ = 1/(α + β). The parameters that describe the C–O transition in Fig. 5 (δ, zδ, γ, zγ), were adjusted to fit the Po–V relationship (see Fig. 6 B) and to reproduce the time course of IK activation at the peak of the Δt–V relationship (+153 mV).
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Figure 3: Voltage dependence of IK delay. (A) The initial time course of IK activation measured at +180 mV from the same patch as Fig. 2 shows a decreased delay following a 1-ms prepulse to various voltages (−80, 0, 40, 80, 100, and 120 mV). To accurately represent the time course of channel opening, currents during the prepulse were scaled by a factor IK(+180)/IK(Vpre) representing the ratio of single channel current amplitudes measured at the test and prepulse voltages. (B) A family of IK evoked at different voltages (+120 to +240 in 20-mV steps, holding potential = −80) are fit with exponential functions (dashed lines), demonstrating a voltage-dependent change in delay. (C) Delay duration (Δt) is plotted versus pulse voltage for two experiments. The plots are fit by functions of the form Δt = 1/(a + b) with a=ao*ezaekt,b=bo*ezbekt(za = +0.28 e, zb = −0.28 e ; (▴) ao = 124 s−1, bo = 4,767 s−1; (•) ao = 98 s−1, bo = 3,902 s−1). The delay was not well determined at the lowest voltages; therefore, fits were constrained with the simplifying assumption za = zb. (D) The average Δt–V relationship (mean ± SEM, n = 6), was obtained after first normalizing individual plots (see text) to mean Δt measured from +180 to +195 mV. The data are fit by the above function (solid line, za = +0.28 e; zb = −0.28 e ; ao = 119 s−1, bo = 4,240 s−1) and reproduced by Fig. 5 [dashed line: zα = +0.28 e, α(0) = 244 s−1, zβ = −0.28 e, β(0) = 8,670 s−1, zδ = 0.155 e, δ(0) = 49.4 s−1, zγ = −0.155 e, γ(0) = 134 s−1]. The rate constants in Fig. 5 that describe voltage sensor movement (α, β) are 2.05-fold greater than (a, b) at all voltages. Thus Δt = 1/(a + b) is proportional the voltage-sensor time constant τ = 1/(α + β). The parameters that describe the C–O transition in Fig. 5 (δ, zδ, γ, zγ), were adjusted to fit the Po–V relationship (see Fig. 6 B) and to reproduce the time course of IK activation at the peak of the Δt–V relationship (+153 mV).

Mentions: It is conceivable that Fig. 5 could give rise to the observed delay kinetics if channels were distributed in states other than C0 at the start of the voltage pulse. This possibility was ruled out by examining the effect of initial conditions on the delay. Fig. 3 A shows the time course of IK evoked at +180 mV after a 1-ms prepulse to voltages between −80 and +120 mV. Prepulses to voltages >0 mV produced a progressive decrease in the delay, resulting in a shift of IK along the time axis analogous to that reported by Cole and Moore 1960 for K+ current in squid axon. A similar effect was reported for hSlo channels by Stefani et al. 1997. This Cole-Moore shift indicates that the initial distribution of channels among closed states is voltage dependent. However, prepulses to −80 or 0 mV had no detectable effect on the delay, suggesting that the closed state distribution does not change at voltages <0 mV. This result supports the assumption that channels mainly occupy the ground state (C0) at −80 mV.


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)

Voltage dependence of IK delay. (A) The initial time course of IK activation measured at +180 mV from the same patch as Fig. 2 shows a decreased delay following a 1-ms prepulse to various voltages (−80, 0, 40, 80, 100, and 120 mV). To accurately represent the time course of channel opening, currents during the prepulse were scaled by a factor IK(+180)/IK(Vpre) representing the ratio of single channel current amplitudes measured at the test and prepulse voltages. (B) A family of IK evoked at different voltages (+120 to +240 in 20-mV steps, holding potential = −80) are fit with exponential functions (dashed lines), demonstrating a voltage-dependent change in delay. (C) Delay duration (Δt) is plotted versus pulse voltage for two experiments. The plots are fit by functions of the form Δt = 1/(a + b) with a=ao*ezaekt,b=bo*ezbekt(za = +0.28 e, zb = −0.28 e ; (▴) ao = 124 s−1, bo = 4,767 s−1; (•) ao = 98 s−1, bo = 3,902 s−1). The delay was not well determined at the lowest voltages; therefore, fits were constrained with the simplifying assumption za = zb. (D) The average Δt–V relationship (mean ± SEM, n = 6), was obtained after first normalizing individual plots (see text) to mean Δt measured from +180 to +195 mV. The data are fit by the above function (solid line, za = +0.28 e; zb = −0.28 e ; ao = 119 s−1, bo = 4,240 s−1) and reproduced by Fig. 5 [dashed line: zα = +0.28 e, α(0) = 244 s−1, zβ = −0.28 e, β(0) = 8,670 s−1, zδ = 0.155 e, δ(0) = 49.4 s−1, zγ = −0.155 e, γ(0) = 134 s−1]. The rate constants in Fig. 5 that describe voltage sensor movement (α, β) are 2.05-fold greater than (a, b) at all voltages. Thus Δt = 1/(a + b) is proportional the voltage-sensor time constant τ = 1/(α + β). The parameters that describe the C–O transition in Fig. 5 (δ, zδ, γ, zγ), were adjusted to fit the Po–V relationship (see Fig. 6 B) and to reproduce the time course of IK activation at the peak of the Δt–V relationship (+153 mV).
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Figure 3: Voltage dependence of IK delay. (A) The initial time course of IK activation measured at +180 mV from the same patch as Fig. 2 shows a decreased delay following a 1-ms prepulse to various voltages (−80, 0, 40, 80, 100, and 120 mV). To accurately represent the time course of channel opening, currents during the prepulse were scaled by a factor IK(+180)/IK(Vpre) representing the ratio of single channel current amplitudes measured at the test and prepulse voltages. (B) A family of IK evoked at different voltages (+120 to +240 in 20-mV steps, holding potential = −80) are fit with exponential functions (dashed lines), demonstrating a voltage-dependent change in delay. (C) Delay duration (Δt) is plotted versus pulse voltage for two experiments. The plots are fit by functions of the form Δt = 1/(a + b) with a=ao*ezaekt,b=bo*ezbekt(za = +0.28 e, zb = −0.28 e ; (▴) ao = 124 s−1, bo = 4,767 s−1; (•) ao = 98 s−1, bo = 3,902 s−1). The delay was not well determined at the lowest voltages; therefore, fits were constrained with the simplifying assumption za = zb. (D) The average Δt–V relationship (mean ± SEM, n = 6), was obtained after first normalizing individual plots (see text) to mean Δt measured from +180 to +195 mV. The data are fit by the above function (solid line, za = +0.28 e; zb = −0.28 e ; ao = 119 s−1, bo = 4,240 s−1) and reproduced by Fig. 5 [dashed line: zα = +0.28 e, α(0) = 244 s−1, zβ = −0.28 e, β(0) = 8,670 s−1, zδ = 0.155 e, δ(0) = 49.4 s−1, zγ = −0.155 e, γ(0) = 134 s−1]. The rate constants in Fig. 5 that describe voltage sensor movement (α, β) are 2.05-fold greater than (a, b) at all voltages. Thus Δt = 1/(a + b) is proportional the voltage-sensor time constant τ = 1/(α + β). The parameters that describe the C–O transition in Fig. 5 (δ, zδ, γ, zγ), were adjusted to fit the Po–V relationship (see Fig. 6 B) and to reproduce the time course of IK activation at the peak of the Δt–V relationship (+153 mV).
Mentions: It is conceivable that Fig. 5 could give rise to the observed delay kinetics if channels were distributed in states other than C0 at the start of the voltage pulse. This possibility was ruled out by examining the effect of initial conditions on the delay. Fig. 3 A shows the time course of IK evoked at +180 mV after a 1-ms prepulse to voltages between −80 and +120 mV. Prepulses to voltages >0 mV produced a progressive decrease in the delay, resulting in a shift of IK along the time axis analogous to that reported by Cole and Moore 1960 for K+ current in squid axon. A similar effect was reported for hSlo channels by Stefani et al. 1997. This Cole-Moore shift indicates that the initial distribution of channels among closed states is voltage dependent. However, prepulses to −80 or 0 mV had no detectable effect on the delay, suggesting that the closed state distribution does not change at voltages <0 mV. This result supports the assumption that channels mainly occupy the ground state (C0) at −80 mV.

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