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Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

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

Bottom Line: These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J.Physiol. 114:277-304).The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.

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
Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.

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Cg represents mSlo charge movement. (A) The Ca2+ sensitivity of Cg is illustrated by comparing Cg–V relationships obtained in 0 or 60 μM [Ca2+]i from the same patch at 868 Hz. Peak Cg in 0 Ca was 39% larger than in 60 Ca2+, but traces were normalized to show the shift in the position of peak Cg along the voltage axis. (B) Cg–V relationships for mSlo wild-type (WT, 1,736 Hz) and mutant (R207Q, 1,500 Hz) channels are similar in shape but are shifted by more than 250 mV relative to each other. Fits, representing the derivative of Boltzmann functions, are superimposed on the data (dashed lines; WT: z = 0.58 e, Vh = 132 mV; R207Q: z = 0.74, Vh = −143 mV). (C) Cg–V relationships for wild-type channels decrease in amplitude as the frequency of the sinusoidal voltage command is increased from 200 to 6,944 Hz. (D) The orthogonal component of the admittance signal (Gg) increases with frequency. (E) DC current measured during the voltage ramp (see Materials and Methods) changes linearly with voltage, demonstrating that the G signal does not represent voltage-dependent changes in Gm and indicating a membrane/seal resistance (125 GV) over the entire voltage range. (F) Cg (solid symbols) and Gg (open symbols) measured at +120 mV are plotted versus frequency for two experiments and are fit by Lorenzian functions, described in the text, with a time constant of 70 μs.
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Figure 2: Cg represents mSlo charge movement. (A) The Ca2+ sensitivity of Cg is illustrated by comparing Cg–V relationships obtained in 0 or 60 μM [Ca2+]i from the same patch at 868 Hz. Peak Cg in 0 Ca was 39% larger than in 60 Ca2+, but traces were normalized to show the shift in the position of peak Cg along the voltage axis. (B) Cg–V relationships for mSlo wild-type (WT, 1,736 Hz) and mutant (R207Q, 1,500 Hz) channels are similar in shape but are shifted by more than 250 mV relative to each other. Fits, representing the derivative of Boltzmann functions, are superimposed on the data (dashed lines; WT: z = 0.58 e, Vh = 132 mV; R207Q: z = 0.74, Vh = −143 mV). (C) Cg–V relationships for wild-type channels decrease in amplitude as the frequency of the sinusoidal voltage command is increased from 200 to 6,944 Hz. (D) The orthogonal component of the admittance signal (Gg) increases with frequency. (E) DC current measured during the voltage ramp (see Materials and Methods) changes linearly with voltage, demonstrating that the G signal does not represent voltage-dependent changes in Gm and indicating a membrane/seal resistance (125 GV) over the entire voltage range. (F) Cg (solid symbols) and Gg (open symbols) measured at +120 mV are plotted versus frequency for two experiments and are fit by Lorenzian functions, described in the text, with a time constant of 70 μs.

Mentions: Two approaches were used to measure mSlo gating charge movement. The first involved clamping the membrane with a sinusoidal voltage command and measuring gating charge as a voltage-dependent component of membrane capacitance using admittance analysis (Fernandez et al. 1982). The second involved conventional measurement of gating currents in response to voltage steps. Although the bulk of the analysis was performed using voltage steps, the admittance analysis is presented briefly first (see Fig. 1 and Fig. 2) to provide an initial characterization of mSlo charge movement and to demonstrate several necessary controls.


Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

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

Cg represents mSlo charge movement. (A) The Ca2+ sensitivity of Cg is illustrated by comparing Cg–V relationships obtained in 0 or 60 μM [Ca2+]i from the same patch at 868 Hz. Peak Cg in 0 Ca was 39% larger than in 60 Ca2+, but traces were normalized to show the shift in the position of peak Cg along the voltage axis. (B) Cg–V relationships for mSlo wild-type (WT, 1,736 Hz) and mutant (R207Q, 1,500 Hz) channels are similar in shape but are shifted by more than 250 mV relative to each other. Fits, representing the derivative of Boltzmann functions, are superimposed on the data (dashed lines; WT: z = 0.58 e, Vh = 132 mV; R207Q: z = 0.74, Vh = −143 mV). (C) Cg–V relationships for wild-type channels decrease in amplitude as the frequency of the sinusoidal voltage command is increased from 200 to 6,944 Hz. (D) The orthogonal component of the admittance signal (Gg) increases with frequency. (E) DC current measured during the voltage ramp (see Materials and Methods) changes linearly with voltage, demonstrating that the G signal does not represent voltage-dependent changes in Gm and indicating a membrane/seal resistance (125 GV) over the entire voltage range. (F) Cg (solid symbols) and Gg (open symbols) measured at +120 mV are plotted versus frequency for two experiments and are fit by Lorenzian functions, described in the text, with a time constant of 70 μs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230644&req=5

Figure 2: Cg represents mSlo charge movement. (A) The Ca2+ sensitivity of Cg is illustrated by comparing Cg–V relationships obtained in 0 or 60 μM [Ca2+]i from the same patch at 868 Hz. Peak Cg in 0 Ca was 39% larger than in 60 Ca2+, but traces were normalized to show the shift in the position of peak Cg along the voltage axis. (B) Cg–V relationships for mSlo wild-type (WT, 1,736 Hz) and mutant (R207Q, 1,500 Hz) channels are similar in shape but are shifted by more than 250 mV relative to each other. Fits, representing the derivative of Boltzmann functions, are superimposed on the data (dashed lines; WT: z = 0.58 e, Vh = 132 mV; R207Q: z = 0.74, Vh = −143 mV). (C) Cg–V relationships for wild-type channels decrease in amplitude as the frequency of the sinusoidal voltage command is increased from 200 to 6,944 Hz. (D) The orthogonal component of the admittance signal (Gg) increases with frequency. (E) DC current measured during the voltage ramp (see Materials and Methods) changes linearly with voltage, demonstrating that the G signal does not represent voltage-dependent changes in Gm and indicating a membrane/seal resistance (125 GV) over the entire voltage range. (F) Cg (solid symbols) and Gg (open symbols) measured at +120 mV are plotted versus frequency for two experiments and are fit by Lorenzian functions, described in the text, with a time constant of 70 μs.
Mentions: Two approaches were used to measure mSlo gating charge movement. The first involved clamping the membrane with a sinusoidal voltage command and measuring gating charge as a voltage-dependent component of membrane capacitance using admittance analysis (Fernandez et al. 1982). The second involved conventional measurement of gating currents in response to voltage steps. Although the bulk of the analysis was performed using voltage steps, the admittance analysis is presented briefly first (see Fig. 1 and Fig. 2) to provide an initial characterization of mSlo charge movement and to demonstrate several necessary controls.

Bottom Line: These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J.Physiol. 114:277-304).The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.

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
Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.

Show MeSH
Related in: MedlinePlus