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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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mSlo gating capacitance. (A) Voltage-dependent membrane capacitance (Cg) is plotted versus voltage for patches excised from HEK 293 cells that expressed mSlo channels (mSlo) or were not transfected (control). Each trace represents the average response to two voltage ramps (see panel C). The baseline (Cg = 0) was set to the mean of Cm between −150 and −100 mV. The Cg–V relationship for mSlo is fit by the derivative of a Boltzmann function with respect to voltage (dashed line) with z = 0.59 e and Vh = 133 mV. (B) The Qg–V relationship, determined by integrating the Cg–V, is fit by a Boltzmann function. The shape of the average GK–V relationship (symbols; from Horrigan et al. 1999) can be approximated by raising the Qg–V fit to the 4th power (Fit4). (C) The voltage protocol used to measure Cg in panel A consists of a 1-s voltage ramp from −160 to +200 mV superimposed with a sinusoidal command (30 mV amplitude, 868 Hz). Capacitance was determined for each period of the sinusoid. (D) Cg–V relationships obtained from channels expressed in a Xenopus oocyte using sinusoidal voltage amplitudes of 3 mV (symbols) or 30 mV (solid line) at 1,736 Hz are superimposable, indicating that sinwave amplitude does not effect the measurement. The 3 and 30 mV traces represent the average response to eight and four voltage ramps, respectively. Data are plotted at 3-mV intervals, representing the mean capacitance over eight periods of the sinusoid. The internal solution did not contain crown ether (18C6TA).
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Figure 1: mSlo gating capacitance. (A) Voltage-dependent membrane capacitance (Cg) is plotted versus voltage for patches excised from HEK 293 cells that expressed mSlo channels (mSlo) or were not transfected (control). Each trace represents the average response to two voltage ramps (see panel C). The baseline (Cg = 0) was set to the mean of Cm between −150 and −100 mV. The Cg–V relationship for mSlo is fit by the derivative of a Boltzmann function with respect to voltage (dashed line) with z = 0.59 e and Vh = 133 mV. (B) The Qg–V relationship, determined by integrating the Cg–V, is fit by a Boltzmann function. The shape of the average GK–V relationship (symbols; from Horrigan et al. 1999) can be approximated by raising the Qg–V fit to the 4th power (Fit4). (C) The voltage protocol used to measure Cg in panel A consists of a 1-s voltage ramp from −160 to +200 mV superimposed with a sinusoidal command (30 mV amplitude, 868 Hz). Capacitance was determined for each period of the sinusoid. (D) Cg–V relationships obtained from channels expressed in a Xenopus oocyte using sinusoidal voltage amplitudes of 3 mV (symbols) or 30 mV (solid line) at 1,736 Hz are superimposable, indicating that sinwave amplitude does not effect the measurement. The 3 and 30 mV traces represent the average response to eight and four voltage ramps, respectively. Data are plotted at 3-mV intervals, representing the mean capacitance over eight periods of the sinusoid. The internal solution did not contain crown ether (18C6TA).

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)

mSlo gating capacitance. (A) Voltage-dependent membrane capacitance (Cg) is plotted versus voltage for patches excised from HEK 293 cells that expressed mSlo channels (mSlo) or were not transfected (control). Each trace represents the average response to two voltage ramps (see panel C). The baseline (Cg = 0) was set to the mean of Cm between −150 and −100 mV. The Cg–V relationship for mSlo is fit by the derivative of a Boltzmann function with respect to voltage (dashed line) with z = 0.59 e and Vh = 133 mV. (B) The Qg–V relationship, determined by integrating the Cg–V, is fit by a Boltzmann function. The shape of the average GK–V relationship (symbols; from Horrigan et al. 1999) can be approximated by raising the Qg–V fit to the 4th power (Fit4). (C) The voltage protocol used to measure Cg in panel A consists of a 1-s voltage ramp from −160 to +200 mV superimposed with a sinusoidal command (30 mV amplitude, 868 Hz). Capacitance was determined for each period of the sinusoid. (D) Cg–V relationships obtained from channels expressed in a Xenopus oocyte using sinusoidal voltage amplitudes of 3 mV (symbols) or 30 mV (solid line) at 1,736 Hz are superimposable, indicating that sinwave amplitude does not effect the measurement. The 3 and 30 mV traces represent the average response to eight and four voltage ramps, respectively. Data are plotted at 3-mV intervals, representing the mean capacitance over eight periods of the sinusoid. The internal solution did not contain crown ether (18C6TA).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230644&req=5

Figure 1: mSlo gating capacitance. (A) Voltage-dependent membrane capacitance (Cg) is plotted versus voltage for patches excised from HEK 293 cells that expressed mSlo channels (mSlo) or were not transfected (control). Each trace represents the average response to two voltage ramps (see panel C). The baseline (Cg = 0) was set to the mean of Cm between −150 and −100 mV. The Cg–V relationship for mSlo is fit by the derivative of a Boltzmann function with respect to voltage (dashed line) with z = 0.59 e and Vh = 133 mV. (B) The Qg–V relationship, determined by integrating the Cg–V, is fit by a Boltzmann function. The shape of the average GK–V relationship (symbols; from Horrigan et al. 1999) can be approximated by raising the Qg–V fit to the 4th power (Fit4). (C) The voltage protocol used to measure Cg in panel A consists of a 1-s voltage ramp from −160 to +200 mV superimposed with a sinusoidal command (30 mV amplitude, 868 Hz). Capacitance was determined for each period of the sinusoid. (D) Cg–V relationships obtained from channels expressed in a Xenopus oocyte using sinusoidal voltage amplitudes of 3 mV (symbols) or 30 mV (solid line) at 1,736 Hz are superimposable, indicating that sinwave amplitude does not effect the measurement. The 3 and 30 mV traces represent the average response to eight and four voltage ramps, respectively. Data are plotted at 3-mV intervals, representing the mean capacitance over eight periods of the sinusoid. The internal solution did not contain crown ether (18C6TA).
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