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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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Simulation of α1G T-currents. Parameters are given in the legend to Fig. 14. Currents were calculated assuming a reversal potential of +25 mV, with a linear open-channel I–V. (A) Currents during 60-ms depolarizations from −100 mV to voltages from −90 to +70 mV, as in Fig. 1 A. Peak current–voltage relations (B), the peak probability that the channel is open (C), and the time to peak (D) from the protocol of A (compare with Fig. 1 B and Fig. 4A and Fig. B, respectively). The smooth curve in C is the sum of two Boltzmann functions: . (E) Currents from the protocol of Fig. 3, recorded in 20-mV increments from −120 to +60 mV, after 2-ms depolarizations to +60 mV. (F) Time constants for inactivation (triangles) from A, and for inactivation plus deactivation (squares) from E, shown as in Fig. 5 A. Time constants changed e-fold for 26 mV from −70 to −120 mV. (G) Currents during a train of 5-ms depolarizations from −100 to −20 mV, given at 15-ms intervals, superimposed on the current during a 50-ms depolarization to −20 mV. The lower panel shows the summed probability of being in an inactivated state, in response to the four depolarizations shown above. Compare with Fig. 12. Note that net inactivation occurs during the tail current after the first two steps, despite some recovery from inactivation (more apparent after the last two steps). (H) Nonmonotonic recovery from inactivation, using the protocol of Fig. 13.
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Figure 15: Simulation of α1G T-currents. Parameters are given in the legend to Fig. 14. Currents were calculated assuming a reversal potential of +25 mV, with a linear open-channel I–V. (A) Currents during 60-ms depolarizations from −100 mV to voltages from −90 to +70 mV, as in Fig. 1 A. Peak current–voltage relations (B), the peak probability that the channel is open (C), and the time to peak (D) from the protocol of A (compare with Fig. 1 B and Fig. 4A and Fig. B, respectively). The smooth curve in C is the sum of two Boltzmann functions: . (E) Currents from the protocol of Fig. 3, recorded in 20-mV increments from −120 to +60 mV, after 2-ms depolarizations to +60 mV. (F) Time constants for inactivation (triangles) from A, and for inactivation plus deactivation (squares) from E, shown as in Fig. 5 A. Time constants changed e-fold for 26 mV from −70 to −120 mV. (G) Currents during a train of 5-ms depolarizations from −100 to −20 mV, given at 15-ms intervals, superimposed on the current during a 50-ms depolarization to −20 mV. The lower panel shows the summed probability of being in an inactivated state, in response to the four depolarizations shown above. Compare with Fig. 12. Note that net inactivation occurs during the tail current after the first two steps, despite some recovery from inactivation (more apparent after the last two steps). (H) Nonmonotonic recovery from inactivation, using the protocol of Fig. 13.

Mentions: The scheme of Fig. 14 A can accurately describe many aspects of the experimental data (Fig. 15). Current records cross over at negative voltages (Fig. 15 A) and activate in the appropriate voltage range (Fig. 15B and Fig. C). The sum of two Boltzmann distributions was required for accurate description of the simulated activation curve (Fig. 15 C; compare with Fig. 4 A). The voltage dependence of the time to peak (Fig. 15 D) resembled the experimental data (Fig. 4 B), approaching 1 ms at strongly positive voltages. Tail currents from the protocol of Fig. 3 A decayed nearly monoexponentially (Fig. 15E and Fig. F), although the model does not describe the small increase in time constant at positive voltages (Fig. 5 A). The model reproduces cumulative inactivation (Fig. 15 G), with considerable inactivation occurring during tail currents. Nonmonotonic recovery from inactivation occurs after brief (5-ms) steps, although this is barely visible in the P3/P1 ratio (Fig. 15 H).


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

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

Simulation of α1G T-currents. Parameters are given in the legend to Fig. 14. Currents were calculated assuming a reversal potential of +25 mV, with a linear open-channel I–V. (A) Currents during 60-ms depolarizations from −100 mV to voltages from −90 to +70 mV, as in Fig. 1 A. Peak current–voltage relations (B), the peak probability that the channel is open (C), and the time to peak (D) from the protocol of A (compare with Fig. 1 B and Fig. 4A and Fig. B, respectively). The smooth curve in C is the sum of two Boltzmann functions: . (E) Currents from the protocol of Fig. 3, recorded in 20-mV increments from −120 to +60 mV, after 2-ms depolarizations to +60 mV. (F) Time constants for inactivation (triangles) from A, and for inactivation plus deactivation (squares) from E, shown as in Fig. 5 A. Time constants changed e-fold for 26 mV from −70 to −120 mV. (G) Currents during a train of 5-ms depolarizations from −100 to −20 mV, given at 15-ms intervals, superimposed on the current during a 50-ms depolarization to −20 mV. The lower panel shows the summed probability of being in an inactivated state, in response to the four depolarizations shown above. Compare with Fig. 12. Note that net inactivation occurs during the tail current after the first two steps, despite some recovery from inactivation (more apparent after the last two steps). (H) Nonmonotonic recovery from inactivation, using the protocol of Fig. 13.
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Figure 15: Simulation of α1G T-currents. Parameters are given in the legend to Fig. 14. Currents were calculated assuming a reversal potential of +25 mV, with a linear open-channel I–V. (A) Currents during 60-ms depolarizations from −100 mV to voltages from −90 to +70 mV, as in Fig. 1 A. Peak current–voltage relations (B), the peak probability that the channel is open (C), and the time to peak (D) from the protocol of A (compare with Fig. 1 B and Fig. 4A and Fig. B, respectively). The smooth curve in C is the sum of two Boltzmann functions: . (E) Currents from the protocol of Fig. 3, recorded in 20-mV increments from −120 to +60 mV, after 2-ms depolarizations to +60 mV. (F) Time constants for inactivation (triangles) from A, and for inactivation plus deactivation (squares) from E, shown as in Fig. 5 A. Time constants changed e-fold for 26 mV from −70 to −120 mV. (G) Currents during a train of 5-ms depolarizations from −100 to −20 mV, given at 15-ms intervals, superimposed on the current during a 50-ms depolarization to −20 mV. The lower panel shows the summed probability of being in an inactivated state, in response to the four depolarizations shown above. Compare with Fig. 12. Note that net inactivation occurs during the tail current after the first two steps, despite some recovery from inactivation (more apparent after the last two steps). (H) Nonmonotonic recovery from inactivation, using the protocol of Fig. 13.
Mentions: The scheme of Fig. 14 A can accurately describe many aspects of the experimental data (Fig. 15). Current records cross over at negative voltages (Fig. 15 A) and activate in the appropriate voltage range (Fig. 15B and Fig. C). The sum of two Boltzmann distributions was required for accurate description of the simulated activation curve (Fig. 15 C; compare with Fig. 4 A). The voltage dependence of the time to peak (Fig. 15 D) resembled the experimental data (Fig. 4 B), approaching 1 ms at strongly positive voltages. Tail currents from the protocol of Fig. 3 A decayed nearly monoexponentially (Fig. 15E and Fig. F), although the model does not describe the small increase in time constant at positive voltages (Fig. 5 A). The model reproduces cumulative inactivation (Fig. 15 G), with considerable inactivation occurring during tail currents. Nonmonotonic recovery from inactivation occurs after brief (5-ms) steps, although this is barely visible in the P3/P1 ratio (Fig. 15 H).

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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Related in: MedlinePlus