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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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Voltage dependence of steady state inactivation. Steps lasting 1 s to the indicated voltages were followed by 5-ms test pulses to −20 mV. The protocol was run either with no preceding depolarization (for inactivation) or immediately after a 60-ms step to −20 mV (for recovery). For the recovery protocol, the values are the ratio of current in the test pulse to the current during the 60-ms prepulse, measured at comparable times. For the inactivation protocol, currents were normalized to the value at −100 mV (i.e., with no prepulse). Test pulses (with no prepulse) were given before and after the rest of the protocol, to control for rundown or accumulation of inactivation. The smooth curves are fits to a Boltzmann relation, with  (inactivation) and  (recovery). Cell a8n02, 1 kHz analogue antialias filtering.
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Figure 7: Voltage dependence of steady state inactivation. Steps lasting 1 s to the indicated voltages were followed by 5-ms test pulses to −20 mV. The protocol was run either with no preceding depolarization (for inactivation) or immediately after a 60-ms step to −20 mV (for recovery). For the recovery protocol, the values are the ratio of current in the test pulse to the current during the 60-ms prepulse, measured at comparable times. For the inactivation protocol, currents were normalized to the value at −100 mV (i.e., with no prepulse). Test pulses (with no prepulse) were given before and after the rest of the protocol, to control for rundown or accumulation of inactivation. The smooth curves are fits to a Boltzmann relation, with (inactivation) and (recovery). Cell a8n02, 1 kHz analogue antialias filtering.

Mentions: Fig. 6 suggests that inactivation should reach a steady state by ∼1 s. To test that, and to measure the properties of steady state inactivation, voltage steps lasting 1 s were given either directly from −100 mV, or after 60-ms steps to −20 mV to inactivate most of the channels (Fig. 7). At steady state, the measured channel availability should depend only on the tested voltage, i.e., the channel should have “forgotten” whether the inactivating pulse to −20 mV had been given. This comparison can only be done in a narrow voltage range, near the midpoint of the steady state inactivation curve, where the amplitudes of inactivation and recovery are both measurable. The two protocols gave almost identical availability curves: . When the voltage steps lasted <1 s, the measured V1/2 was more negative for the recovery protocol than for inactivation, demonstrating that steady state had not been reached (data not shown).


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

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

Voltage dependence of steady state inactivation. Steps lasting 1 s to the indicated voltages were followed by 5-ms test pulses to −20 mV. The protocol was run either with no preceding depolarization (for inactivation) or immediately after a 60-ms step to −20 mV (for recovery). For the recovery protocol, the values are the ratio of current in the test pulse to the current during the 60-ms prepulse, measured at comparable times. For the inactivation protocol, currents were normalized to the value at −100 mV (i.e., with no prepulse). Test pulses (with no prepulse) were given before and after the rest of the protocol, to control for rundown or accumulation of inactivation. The smooth curves are fits to a Boltzmann relation, with  (inactivation) and  (recovery). Cell a8n02, 1 kHz analogue antialias filtering.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: Voltage dependence of steady state inactivation. Steps lasting 1 s to the indicated voltages were followed by 5-ms test pulses to −20 mV. The protocol was run either with no preceding depolarization (for inactivation) or immediately after a 60-ms step to −20 mV (for recovery). For the recovery protocol, the values are the ratio of current in the test pulse to the current during the 60-ms prepulse, measured at comparable times. For the inactivation protocol, currents were normalized to the value at −100 mV (i.e., with no prepulse). Test pulses (with no prepulse) were given before and after the rest of the protocol, to control for rundown or accumulation of inactivation. The smooth curves are fits to a Boltzmann relation, with (inactivation) and (recovery). Cell a8n02, 1 kHz analogue antialias filtering.
Mentions: Fig. 6 suggests that inactivation should reach a steady state by ∼1 s. To test that, and to measure the properties of steady state inactivation, voltage steps lasting 1 s were given either directly from −100 mV, or after 60-ms steps to −20 mV to inactivate most of the channels (Fig. 7). At steady state, the measured channel availability should depend only on the tested voltage, i.e., the channel should have “forgotten” whether the inactivating pulse to −20 mV had been given. This comparison can only be done in a narrow voltage range, near the midpoint of the steady state inactivation curve, where the amplitudes of inactivation and recovery are both measurable. The two protocols gave almost identical availability curves: . When the voltage steps lasted <1 s, the measured V1/2 was more negative for the recovery protocol than for inactivation, demonstrating that steady state had not been reached (data not shown).

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