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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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Instantaneous current–voltage relations for α1G channels. (A) Sample current records, with 5 kHz Gaussian filtering, from cell e8612. The initial step to +60 mV lasted 2 ms. Records are shown for repolarizations in 20-mV increments. 2-ms test pulses to +60 mV were given at the end of the protocol, 20-ms after repolarization to −100 mV, to assess inactivation (not shown; see Fig. 11 below). (B) Instantaneous current–voltage relations, measured from the protocol of A, averaged from eight cells. Currents were measured at the peak of the tail current in each cell, which varied from 0.3 to 0.5 ms in these cells.
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Figure 3: Instantaneous current–voltage relations for α1G channels. (A) Sample current records, with 5 kHz Gaussian filtering, from cell e8612. The initial step to +60 mV lasted 2 ms. Records are shown for repolarizations in 20-mV increments. 2-ms test pulses to +60 mV were given at the end of the protocol, 20-ms after repolarization to −100 mV, to assess inactivation (not shown; see Fig. 11 below). (B) Instantaneous current–voltage relations, measured from the protocol of A, averaged from eight cells. Currents were measured at the peak of the tail current in each cell, which varied from 0.3 to 0.5 ms in these cells.

Mentions: Since the currents recorded could be >1 nA, data were examined closely for signs of series resistance error. Clamp speed was assessed by the rise time of tail currents, and steady state accuracy by the effect of partial inactivation on the time course of tail currents. For cells used for kinetic analysis of tail currents (e.g., Fig. 3), the 10–90% rise time was 0.15–0.35 ms after 10-kHz analogue filtering. Prepulses that caused ∼70% inactivation (using the protocol illustrated in the inset to Fig. 11) affected the time constant for deactivation at −100 mV by ≤15%. Since the measured time constants changed 37% per 10 mV near −100 mV (see Fig. 5), this suggests ≤5 mV error.


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

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

Instantaneous current–voltage relations for α1G channels. (A) Sample current records, with 5 kHz Gaussian filtering, from cell e8612. The initial step to +60 mV lasted 2 ms. Records are shown for repolarizations in 20-mV increments. 2-ms test pulses to +60 mV were given at the end of the protocol, 20-ms after repolarization to −100 mV, to assess inactivation (not shown; see Fig. 11 below). (B) Instantaneous current–voltage relations, measured from the protocol of A, averaged from eight cells. Currents were measured at the peak of the tail current in each cell, which varied from 0.3 to 0.5 ms in these cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Instantaneous current–voltage relations for α1G channels. (A) Sample current records, with 5 kHz Gaussian filtering, from cell e8612. The initial step to +60 mV lasted 2 ms. Records are shown for repolarizations in 20-mV increments. 2-ms test pulses to +60 mV were given at the end of the protocol, 20-ms after repolarization to −100 mV, to assess inactivation (not shown; see Fig. 11 below). (B) Instantaneous current–voltage relations, measured from the protocol of A, averaged from eight cells. Currents were measured at the peak of the tail current in each cell, which varied from 0.3 to 0.5 ms in these cells.
Mentions: Since the currents recorded could be >1 nA, data were examined closely for signs of series resistance error. Clamp speed was assessed by the rise time of tail currents, and steady state accuracy by the effect of partial inactivation on the time course of tail currents. For cells used for kinetic analysis of tail currents (e.g., Fig. 3), the 10–90% rise time was 0.15–0.35 ms after 10-kHz analogue filtering. Prepulses that caused ∼70% inactivation (using the protocol illustrated in the inset to Fig. 11) affected the time constant for deactivation at −100 mV by ≤15%. Since the measured time constants changed 37% per 10 mV near −100 mV (see Fig. 5), this suggests ≤5 mV error.

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