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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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Kinetics of IK delay. (A1) IK evoked at +180 mV (7°C) is compared with the prediction of a Hodgkin-Huxley model (Scheme III: α = 375 s−1, β = 660 s−1) that approximates the delay in IK (A2), but does not reproduce the subsequent time course of activation. Fig. 5 fits both the delay and activation time course (α = 2,018 s−1, β = 1,172 s−1, δ = 341 s−1, γ = 136 s−1). Both models were constrained to reproduce the steady state open probability measured at the end of the pulse (Po ≅ GK/GKmax = 0.29) and assume channels occupy the first closed state (C0) at the start of the pulse. The derivative of the trace in A [d(IK)/dt] is plotted on linear (B1) and log–log (B2) scales and is fit by a function (1 − e−t/τ)n, where n = 4 and τ = 270 μs (B1 and B2, solid line). A better fit is obtained with n = 2.9 and τ = 316 μs (B2, solid line). The predictions of sequential gating Fig. 5 and Fig. 6 are indicated by dashed lines. (Scheme VI: X = 3, α = 600 s−1, β = 349 s−1,δ = 341 s−1, γ = 136 s−1). Current traces were shifted along the time axis by −25 μs to correct for the instrumentation delay (see methods).
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Figure 2: Kinetics of IK delay. (A1) IK evoked at +180 mV (7°C) is compared with the prediction of a Hodgkin-Huxley model (Scheme III: α = 375 s−1, β = 660 s−1) that approximates the delay in IK (A2), but does not reproduce the subsequent time course of activation. Fig. 5 fits both the delay and activation time course (α = 2,018 s−1, β = 1,172 s−1, δ = 341 s−1, γ = 136 s−1). Both models were constrained to reproduce the steady state open probability measured at the end of the pulse (Po ≅ GK/GKmax = 0.29) and assume channels occupy the first closed state (C0) at the start of the pulse. The derivative of the trace in A [d(IK)/dt] is plotted on linear (B1) and log–log (B2) scales and is fit by a function (1 − e−t/τ)n, where n = 4 and τ = 270 μs (B1 and B2, solid line). A better fit is obtained with n = 2.9 and τ = 316 μs (B2, solid line). The predictions of sequential gating Fig. 5 and Fig. 6 are indicated by dashed lines. (Scheme VI: X = 3, α = 600 s−1, β = 349 s−1,δ = 341 s−1, γ = 136 s−1). Current traces were shifted along the time axis by −25 μs to correct for the instrumentation delay (see methods).

Mentions: Fig. 3 predicts a delay, but it cannot reproduce the kinetics of mSlo activation. The Hodgkin-Huxley model produces an activation time course that is highly sigmoidal because the delay and subsequent activation of IK are both determined by a single process (subunit activation) and therefore occur on a similar time scale. When Fig. 3 is fit to the brief delay in mSlo IK, it predicts an activation time course that is too rapid (Fig. 2, A1 and A2).


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)

Kinetics of IK delay. (A1) IK evoked at +180 mV (7°C) is compared with the prediction of a Hodgkin-Huxley model (Scheme III: α = 375 s−1, β = 660 s−1) that approximates the delay in IK (A2), but does not reproduce the subsequent time course of activation. Fig. 5 fits both the delay and activation time course (α = 2,018 s−1, β = 1,172 s−1, δ = 341 s−1, γ = 136 s−1). Both models were constrained to reproduce the steady state open probability measured at the end of the pulse (Po ≅ GK/GKmax = 0.29) and assume channels occupy the first closed state (C0) at the start of the pulse. The derivative of the trace in A [d(IK)/dt] is plotted on linear (B1) and log–log (B2) scales and is fit by a function (1 − e−t/τ)n, where n = 4 and τ = 270 μs (B1 and B2, solid line). A better fit is obtained with n = 2.9 and τ = 316 μs (B2, solid line). The predictions of sequential gating Fig. 5 and Fig. 6 are indicated by dashed lines. (Scheme VI: X = 3, α = 600 s−1, β = 349 s−1,δ = 341 s−1, γ = 136 s−1). Current traces were shifted along the time axis by −25 μs to correct for the instrumentation delay (see methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230643&req=5

Figure 2: Kinetics of IK delay. (A1) IK evoked at +180 mV (7°C) is compared with the prediction of a Hodgkin-Huxley model (Scheme III: α = 375 s−1, β = 660 s−1) that approximates the delay in IK (A2), but does not reproduce the subsequent time course of activation. Fig. 5 fits both the delay and activation time course (α = 2,018 s−1, β = 1,172 s−1, δ = 341 s−1, γ = 136 s−1). Both models were constrained to reproduce the steady state open probability measured at the end of the pulse (Po ≅ GK/GKmax = 0.29) and assume channels occupy the first closed state (C0) at the start of the pulse. The derivative of the trace in A [d(IK)/dt] is plotted on linear (B1) and log–log (B2) scales and is fit by a function (1 − e−t/τ)n, where n = 4 and τ = 270 μs (B1 and B2, solid line). A better fit is obtained with n = 2.9 and τ = 316 μs (B2, solid line). The predictions of sequential gating Fig. 5 and Fig. 6 are indicated by dashed lines. (Scheme VI: X = 3, α = 600 s−1, β = 349 s−1,δ = 341 s−1, γ = 136 s−1). Current traces were shifted along the time axis by −25 μs to correct for the instrumentation delay (see methods).
Mentions: Fig. 3 predicts a delay, but it cannot reproduce the kinetics of mSlo activation. The Hodgkin-Huxley model produces an activation time course that is highly sigmoidal because the delay and subsequent activation of IK are both determined by a single process (subunit activation) and therefore occur on a similar time scale. When Fig. 3 is fit to the brief delay in mSlo IK, it predicts an activation time course that is too rapid (Fig. 2, A1 and A2).

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