Limits...
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.

Show MeSH

Related in: MedlinePlus

Delay in IK activation. (A1) IK evoked by a voltage pulse to +160 mV from a holding potential of −80 mV at 20°C, representing the average response to 110 pulses. The time course of activation and deactivation are fit by exponential functions (dashed lines). (A2) The record from A1 is plotted on an expanded time scale, showing a delay before IK achieves an exponential time course (dashed line). The delay duration (Δt) is defined as the time where the exponential fit intersects the time axis, and was determined after shifting the IK trace along the time axis by −25 μs to correct for instrumentation delay (see methods). (B1) A family of IK evoked at 5°C in response to 70-ms voltage pulses (+80 to +200 mV in 20-mV steps). Exponential fits (solid lines) are superimposed on the current traces. (B2) The initial activation of IK from B1 exhibits a clear delay. An arrow indicates the start of the voltage pulse. A capacitive transient (control) evoked in response to a pulse to −180 mV was recorded using fast capacity compensation, but no leak subtraction, demonstrating that membrane voltage settles rapidly.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230643&req=5

Figure 1: Delay in IK activation. (A1) IK evoked by a voltage pulse to +160 mV from a holding potential of −80 mV at 20°C, representing the average response to 110 pulses. The time course of activation and deactivation are fit by exponential functions (dashed lines). (A2) The record from A1 is plotted on an expanded time scale, showing a delay before IK achieves an exponential time course (dashed line). The delay duration (Δt) is defined as the time where the exponential fit intersects the time axis, and was determined after shifting the IK trace along the time axis by −25 μs to correct for instrumentation delay (see methods). (B1) A family of IK evoked at 5°C in response to 70-ms voltage pulses (+80 to +200 mV in 20-mV steps). Exponential fits (solid lines) are superimposed on the current traces. (B2) The initial activation of IK from B1 exhibits a clear delay. An arrow indicates the start of the voltage pulse. A capacitive transient (control) evoked in response to a pulse to −180 mV was recorded using fast capacity compensation, but no leak subtraction, demonstrating that membrane voltage settles rapidly.

Mentions: In the absence of Ca2+, mSlo Ca2+-activated K+ channels open in response to membrane depolarization exhibiting a steady state half-activation voltage of approximately +190 mV (Cox et al. 1997a; Cui et al. 1997). Fig. 1 A1 shows mSlo IK evoked in response to a 20-ms pulse to +160 mV from a holding potential of −80 mV in 0 Ca2+ (20°C). The time course of activation and deactivation are well fit by exponential functions (Fig. 1 A1, dashed lines). Similar exponential kinetics are observed over a wide range of voltage and [Ca2+]i (Cui et al. 1997), suggesting a two-state model with a single voltage-dependent transition between a closed and an open state. However, the exponential activation of IK is preceded by a brief delay in the presence or absence of Ca2+ (Cox et al. 1997a; Stefani et al. 1997). Fig. 1 A2 shows the initial time course of IK activation on an expanded time scale. There is a delay of ∼100 μs before the current begins to increase, and at least 300 μs is required to achieve an exponential time course (Fig. 1 A2, dashed line). Although this delay is brief compared with the subsequent relaxation of IK, it is inconsistent with a two-state gating scheme and suggests that mSlo channels undergo one or more transitions among closed states before opening. To better study these rapid transitions, we examined IK activation at a reduced temperature. A family of IK evoked by membrane depolarization at 5°C still exhibits activation kinetics that are well fit by single exponential functions (Fig. 1 B1). The activation is slowed relative to 20°C, and the delay is similarly prolonged (Fig. 1 B2). IK begins to increase after 250 μs and attains an exponential time course after 1 ms. A control trace evoked in response to a voltage pulse to −180 mV is also shown in Fig. 1 B2; the capacitive transient decays within 30 μs, showing that voltage clamp speed and filter properties contribute little to the delay.


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)

Delay in IK activation. (A1) IK evoked by a voltage pulse to +160 mV from a holding potential of −80 mV at 20°C, representing the average response to 110 pulses. The time course of activation and deactivation are fit by exponential functions (dashed lines). (A2) The record from A1 is plotted on an expanded time scale, showing a delay before IK achieves an exponential time course (dashed line). The delay duration (Δt) is defined as the time where the exponential fit intersects the time axis, and was determined after shifting the IK trace along the time axis by −25 μs to correct for instrumentation delay (see methods). (B1) A family of IK evoked at 5°C in response to 70-ms voltage pulses (+80 to +200 mV in 20-mV steps). Exponential fits (solid lines) are superimposed on the current traces. (B2) The initial activation of IK from B1 exhibits a clear delay. An arrow indicates the start of the voltage pulse. A capacitive transient (control) evoked in response to a pulse to −180 mV was recorded using fast capacity compensation, but no leak subtraction, demonstrating that membrane voltage settles rapidly.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230643&req=5

Figure 1: Delay in IK activation. (A1) IK evoked by a voltage pulse to +160 mV from a holding potential of −80 mV at 20°C, representing the average response to 110 pulses. The time course of activation and deactivation are fit by exponential functions (dashed lines). (A2) The record from A1 is plotted on an expanded time scale, showing a delay before IK achieves an exponential time course (dashed line). The delay duration (Δt) is defined as the time where the exponential fit intersects the time axis, and was determined after shifting the IK trace along the time axis by −25 μs to correct for instrumentation delay (see methods). (B1) A family of IK evoked at 5°C in response to 70-ms voltage pulses (+80 to +200 mV in 20-mV steps). Exponential fits (solid lines) are superimposed on the current traces. (B2) The initial activation of IK from B1 exhibits a clear delay. An arrow indicates the start of the voltage pulse. A capacitive transient (control) evoked in response to a pulse to −180 mV was recorded using fast capacity compensation, but no leak subtraction, demonstrating that membrane voltage settles rapidly.
Mentions: In the absence of Ca2+, mSlo Ca2+-activated K+ channels open in response to membrane depolarization exhibiting a steady state half-activation voltage of approximately +190 mV (Cox et al. 1997a; Cui et al. 1997). Fig. 1 A1 shows mSlo IK evoked in response to a 20-ms pulse to +160 mV from a holding potential of −80 mV in 0 Ca2+ (20°C). The time course of activation and deactivation are well fit by exponential functions (Fig. 1 A1, dashed lines). Similar exponential kinetics are observed over a wide range of voltage and [Ca2+]i (Cui et al. 1997), suggesting a two-state model with a single voltage-dependent transition between a closed and an open state. However, the exponential activation of IK is preceded by a brief delay in the presence or absence of Ca2+ (Cox et al. 1997a; Stefani et al. 1997). Fig. 1 A2 shows the initial time course of IK activation on an expanded time scale. There is a delay of ∼100 μs before the current begins to increase, and at least 300 μs is required to achieve an exponential time course (Fig. 1 A2, dashed line). Although this delay is brief compared with the subsequent relaxation of IK, it is inconsistent with a two-state gating scheme and suggests that mSlo channels undergo one or more transitions among closed states before opening. To better study these rapid transitions, we examined IK activation at a reduced temperature. A family of IK evoked by membrane depolarization at 5°C still exhibits activation kinetics that are well fit by single exponential functions (Fig. 1 B1). The activation is slowed relative to 20°C, and the delay is similarly prolonged (Fig. 1 B2). IK begins to increase after 250 μs and attains an exponential time course after 1 ms. A control trace evoked in response to a voltage pulse to −180 mV is also shown in Fig. 1 B2; the capacitive transient decays within 30 μs, showing that voltage clamp speed and filter properties contribute little to the delay.

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