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State-dependent inactivation of the alpha1G T-type calcium channel.

Serrano JR, Perez-Reyes E, Jones SW - J. Gen. Physiol. (1999)

Bottom Line: Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation.The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate.One consequence of state-dependent inactivation is that alpha1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

ABSTRACT
We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with

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Activation of α1G channels. (A) Activation curve, from current ratios: the peak current recorded during depolarization directly to the indicated voltage (the protocol of Fig. 1), divided by the instantaneous current recorded after repolarization from a 2-ms step to +60 mV (the protocol of Fig. 3). Data are not shown for voltages near the reversal potential.  . The smooth curve is a fit to a Boltzmann functions:  mV, e-fold for 9.4 mV, amplitude 0.80. (B) Time to peak, for the protocol of Fig. 1, .
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Figure 4: Activation of α1G channels. (A) Activation curve, from current ratios: the peak current recorded during depolarization directly to the indicated voltage (the protocol of Fig. 1), divided by the instantaneous current recorded after repolarization from a 2-ms step to +60 mV (the protocol of Fig. 3). Data are not shown for voltages near the reversal potential. . The smooth curve is a fit to a Boltzmann functions: mV, e-fold for 9.4 mV, amplitude 0.80. (B) Time to peak, for the protocol of Fig. 1, .

Mentions: Division of the I–V curve (Fig. 1 B) by the instantaneous I–V curve (Fig. 3 B) was used to evaluate the voltage dependence of activation of α1G channels (Fig. 4 A). That ratio (PO,r) should be proportional to the number of channels open at the time of peak current at each voltage. Compared with the usual procedure of measuring tail current amplitudes after depolarizations of fixed duration, this method has the advantage of measuring activation at the maximal value for each voltage. The data at ≤0 mV were fitted to a single Boltzmann function, with half-maximal activation at −48 mV. The data deviate from that function at positive voltages, in part because the current ratios become discontinuous at the reversal potential, but the measured activation was consistently ∼20% greater near +60 mV than near 0 mV. For a rapidly inactivating channel, some channels will inactivate before the point of peak inward current, and the extent of that “hidden” inactivation may vary with voltage. Therefore, the activation curve (Fig. 4 A) should be considered an empirical measurement, which may not fully describe the true voltage dependence of the microscopic activation process.


State-dependent inactivation of the alpha1G T-type calcium channel.

Serrano JR, Perez-Reyes E, Jones SW - J. Gen. Physiol. (1999)

Activation of α1G channels. (A) Activation curve, from current ratios: the peak current recorded during depolarization directly to the indicated voltage (the protocol of Fig. 1), divided by the instantaneous current recorded after repolarization from a 2-ms step to +60 mV (the protocol of Fig. 3). Data are not shown for voltages near the reversal potential.  . The smooth curve is a fit to a Boltzmann functions:  mV, e-fold for 9.4 mV, amplitude 0.80. (B) Time to peak, for the protocol of Fig. 1, .
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230639&req=5

Figure 4: Activation of α1G channels. (A) Activation curve, from current ratios: the peak current recorded during depolarization directly to the indicated voltage (the protocol of Fig. 1), divided by the instantaneous current recorded after repolarization from a 2-ms step to +60 mV (the protocol of Fig. 3). Data are not shown for voltages near the reversal potential. . The smooth curve is a fit to a Boltzmann functions: mV, e-fold for 9.4 mV, amplitude 0.80. (B) Time to peak, for the protocol of Fig. 1, .
Mentions: Division of the I–V curve (Fig. 1 B) by the instantaneous I–V curve (Fig. 3 B) was used to evaluate the voltage dependence of activation of α1G channels (Fig. 4 A). That ratio (PO,r) should be proportional to the number of channels open at the time of peak current at each voltage. Compared with the usual procedure of measuring tail current amplitudes after depolarizations of fixed duration, this method has the advantage of measuring activation at the maximal value for each voltage. The data at ≤0 mV were fitted to a single Boltzmann function, with half-maximal activation at −48 mV. The data deviate from that function at positive voltages, in part because the current ratios become discontinuous at the reversal potential, but the measured activation was consistently ∼20% greater near +60 mV than near 0 mV. For a rapidly inactivating channel, some channels will inactivate before the point of peak inward current, and the extent of that “hidden” inactivation may vary with voltage. Therefore, the activation curve (Fig. 4 A) should be considered an empirical measurement, which may not fully describe the true voltage dependence of the microscopic activation process.

Bottom Line: Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation.The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate.One consequence of state-dependent inactivation is that alpha1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

ABSTRACT
We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with

Show MeSH
Related in: MedlinePlus