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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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The voltage dependence of τ(IK). (A) Time constants [τ(IK)] from the fits in Fig. 4A and Fig. B, are plotted versus voltage and fit (dashed line) by two exponential functions with the indicated equivalent charge (z). The prediction of a two-state model [τ(IK) = 1/(a + b), a=ao*ezaekt,b=bo*ezbekt] is indicated by a solid line (za = 0.37 e, zb = 0.67 e ; ao = 4.86 s−1, bo = 1,323 s−1). (B) τ(IK) is plotted on a log scale versus voltage for all the records in Fig. 4. Data were shifted along the voltage axis by ΔVh = +5.6 mV (see methods). Three regions of exponential voltage dependence are shown by dashed lines with the indicated equivalent charges (z). A solid curve indicates a fit to Fig. 9 (Table , Patch 1). (C) τ(IK)–V plots obtained from multiple experiments at 5° and 20°C (○) were normalized to mean τ(IK) at −80 mV, and then averaged in 15-mV bins (•). Solid curves indicate fits of Fig. 9 to the averaged data (Table : average 5° and 20°C). The dashed curve represents a fit of Fig. 8 to the average 5°C data for V < +100 mV (zβ1 = −0.45 e, β1(0) = 2,400 s−1, zβ2 = −0.14 e, β2(0) = 700 s−1, zα2 = −0.26 e, α2(0) = 300 s−1).
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Figure 5: The voltage dependence of τ(IK). (A) Time constants [τ(IK)] from the fits in Fig. 4A and Fig. B, are plotted versus voltage and fit (dashed line) by two exponential functions with the indicated equivalent charge (z). The prediction of a two-state model [τ(IK) = 1/(a + b), a=ao*ezaekt,b=bo*ezbekt] is indicated by a solid line (za = 0.37 e, zb = 0.67 e ; ao = 4.86 s−1, bo = 1,323 s−1). (B) τ(IK) is plotted on a log scale versus voltage for all the records in Fig. 4. Data were shifted along the voltage axis by ΔVh = +5.6 mV (see methods). Three regions of exponential voltage dependence are shown by dashed lines with the indicated equivalent charges (z). A solid curve indicates a fit to Fig. 9 (Table , Patch 1). (C) τ(IK)–V plots obtained from multiple experiments at 5° and 20°C (○) were normalized to mean τ(IK) at −80 mV, and then averaged in 15-mV bins (•). Solid curves indicate fits of Fig. 9 to the averaged data (Table : average 5° and 20°C). The dashed curve represents a fit of Fig. 8 to the average 5°C data for V < +100 mV (zβ1 = −0.45 e, β1(0) = 2,400 s−1, zβ2 = −0.14 e, β2(0) = 700 s−1, zα2 = −0.26 e, α2(0) = 300 s−1).

Mentions: The voltage dependence of τ(IK) was examined in an experiment illustrated in Fig. 4. IK was activated by stepping from a holding potential of −80 mV to voltages between +100 and +240 mV (Fig. 4 A). IK tail currents were recorded at more negative voltages, following a 50-ms depolarization to +120 mV (Fig. 4, B–D). In all cases, the time course of IK was well fit by an exponential function after a brief delay (Fig. 4, solid lines). τ(IK) is plotted from +30 to +240 mV in Fig. 5 A and exhibits a bell-shaped voltage dependence that can be fit by a two-state model (solid curve) (Cui et al. 1997). This behavior also appears consistent with the prediction of Fig. 5 because τ(IK) increases exponentially with voltage from +30 to +110 mV and decreases exponentially from +180 to +240 (Fig. 5 A, dashed lines) as if τ(IK) is determined by single voltage-dependent rate constants at these voltages. However, our analysis of the delay in IK activation suggests that the voltage range in Fig. 5 A is insufficient to observe the limiting voltage dependence of τ(IK). The Cole-Moore shift (Fig. 3 A) indicates that closed-state equilibria change from +40 to +120 mV, and the weak voltage dependence of the delay (Fig. 3 C) suggests that these equilibria continue to change over a large voltage range.


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)

The voltage dependence of τ(IK). (A) Time constants [τ(IK)] from the fits in Fig. 4A and Fig. B, are plotted versus voltage and fit (dashed line) by two exponential functions with the indicated equivalent charge (z). The prediction of a two-state model [τ(IK) = 1/(a + b), a=ao*ezaekt,b=bo*ezbekt] is indicated by a solid line (za = 0.37 e, zb = 0.67 e ; ao = 4.86 s−1, bo = 1,323 s−1). (B) τ(IK) is plotted on a log scale versus voltage for all the records in Fig. 4. Data were shifted along the voltage axis by ΔVh = +5.6 mV (see methods). Three regions of exponential voltage dependence are shown by dashed lines with the indicated equivalent charges (z). A solid curve indicates a fit to Fig. 9 (Table , Patch 1). (C) τ(IK)–V plots obtained from multiple experiments at 5° and 20°C (○) were normalized to mean τ(IK) at −80 mV, and then averaged in 15-mV bins (•). Solid curves indicate fits of Fig. 9 to the averaged data (Table : average 5° and 20°C). The dashed curve represents a fit of Fig. 8 to the average 5°C data for V < +100 mV (zβ1 = −0.45 e, β1(0) = 2,400 s−1, zβ2 = −0.14 e, β2(0) = 700 s−1, zα2 = −0.26 e, α2(0) = 300 s−1).
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Figure 5: The voltage dependence of τ(IK). (A) Time constants [τ(IK)] from the fits in Fig. 4A and Fig. B, are plotted versus voltage and fit (dashed line) by two exponential functions with the indicated equivalent charge (z). The prediction of a two-state model [τ(IK) = 1/(a + b), a=ao*ezaekt,b=bo*ezbekt] is indicated by a solid line (za = 0.37 e, zb = 0.67 e ; ao = 4.86 s−1, bo = 1,323 s−1). (B) τ(IK) is plotted on a log scale versus voltage for all the records in Fig. 4. Data were shifted along the voltage axis by ΔVh = +5.6 mV (see methods). Three regions of exponential voltage dependence are shown by dashed lines with the indicated equivalent charges (z). A solid curve indicates a fit to Fig. 9 (Table , Patch 1). (C) τ(IK)–V plots obtained from multiple experiments at 5° and 20°C (○) were normalized to mean τ(IK) at −80 mV, and then averaged in 15-mV bins (•). Solid curves indicate fits of Fig. 9 to the averaged data (Table : average 5° and 20°C). The dashed curve represents a fit of Fig. 8 to the average 5°C data for V < +100 mV (zβ1 = −0.45 e, β1(0) = 2,400 s−1, zβ2 = −0.14 e, β2(0) = 700 s−1, zα2 = −0.26 e, α2(0) = 300 s−1).
Mentions: The voltage dependence of τ(IK) was examined in an experiment illustrated in Fig. 4. IK was activated by stepping from a holding potential of −80 mV to voltages between +100 and +240 mV (Fig. 4 A). IK tail currents were recorded at more negative voltages, following a 50-ms depolarization to +120 mV (Fig. 4, B–D). In all cases, the time course of IK was well fit by an exponential function after a brief delay (Fig. 4, solid lines). τ(IK) is plotted from +30 to +240 mV in Fig. 5 A and exhibits a bell-shaped voltage dependence that can be fit by a two-state model (solid curve) (Cui et al. 1997). This behavior also appears consistent with the prediction of Fig. 5 because τ(IK) increases exponentially with voltage from +30 to +110 mV and decreases exponentially from +180 to +240 (Fig. 5 A, dashed lines) as if τ(IK) is determined by single voltage-dependent rate constants at these voltages. However, our analysis of the delay in IK activation suggests that the voltage range in Fig. 5 A is insufficient to observe the limiting voltage dependence of τ(IK). The Cole-Moore shift (Fig. 3 A) indicates that closed-state equilibria change from +40 to +120 mV, and the weak voltage dependence of the delay (Fig. 3 C) suggests that these equilibria continue to change over a large voltage range.

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