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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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Test for window current. (A) Incomplete inactivation after 120-ms depolarizations. Tail currents at −100 mV were fitted to a single exponential (plus a constant) beginning 0.6–0.8 ms after repolarization from the voltages indicated (protocol of Fig. 1, except voltage steps lasted 120 ms). PO,r was calculated by dividing the initial tail current amplitude (sum of the exponentially decaying component, plus the constant) by the instantaneous current at −100 mV after a 2-ms depolarization to +60 mV (the protocol of Fig. 3). Each symbol is a different cell , and the line is drawn through the mean values. (B) Incomplete inactivation for 300-ms depolarizations to −20 mV. The records are the averaged currents from four depolarizations, in each of five cells. The inset shows the current at the end of the step and the tail current, at a 10× higher amplification. 1 kHz Gaussian filter.
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Figure 8: Test for window current. (A) Incomplete inactivation after 120-ms depolarizations. Tail currents at −100 mV were fitted to a single exponential (plus a constant) beginning 0.6–0.8 ms after repolarization from the voltages indicated (protocol of Fig. 1, except voltage steps lasted 120 ms). PO,r was calculated by dividing the initial tail current amplitude (sum of the exponentially decaying component, plus the constant) by the instantaneous current at −100 mV after a 2-ms depolarization to +60 mV (the protocol of Fig. 3). Each symbol is a different cell , and the line is drawn through the mean values. (B) Incomplete inactivation for 300-ms depolarizations to −20 mV. The records are the averaged currents from four depolarizations, in each of five cells. The inset shows the current at the end of the step and the tail current, at a 10× higher amplification. 1 kHz Gaussian filter.

Mentions: The inactivation curve could be described well by a single Boltzmann relation, assuming that channels inactivate fully at depolarized voltages (Fig. 7). The currents recorded during depolarizations do decay to near zero, but small currents are consistently observed at the end of the pulse (Fig. 1 A). This was observed even after depolarizations lasting 120 ms (Fig. 8 A). If the inactivated state is fully absorbing, only 0.0003 of the channels should remain open after 120 ms , but the peak tail current amplitudes correspond to PO,r ∼ 0.02 over a wide voltage range (−60 to +70 mV). The tail currents were small and noisy, so the measured current amplitudes show considerable variability, but residual channel activation was clearly detectable.


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

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

Test for window current. (A) Incomplete inactivation after 120-ms depolarizations. Tail currents at −100 mV were fitted to a single exponential (plus a constant) beginning 0.6–0.8 ms after repolarization from the voltages indicated (protocol of Fig. 1, except voltage steps lasted 120 ms). PO,r was calculated by dividing the initial tail current amplitude (sum of the exponentially decaying component, plus the constant) by the instantaneous current at −100 mV after a 2-ms depolarization to +60 mV (the protocol of Fig. 3). Each symbol is a different cell , and the line is drawn through the mean values. (B) Incomplete inactivation for 300-ms depolarizations to −20 mV. The records are the averaged currents from four depolarizations, in each of five cells. The inset shows the current at the end of the step and the tail current, at a 10× higher amplification. 1 kHz Gaussian filter.
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Related In: Results  -  Collection

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Figure 8: Test for window current. (A) Incomplete inactivation after 120-ms depolarizations. Tail currents at −100 mV were fitted to a single exponential (plus a constant) beginning 0.6–0.8 ms after repolarization from the voltages indicated (protocol of Fig. 1, except voltage steps lasted 120 ms). PO,r was calculated by dividing the initial tail current amplitude (sum of the exponentially decaying component, plus the constant) by the instantaneous current at −100 mV after a 2-ms depolarization to +60 mV (the protocol of Fig. 3). Each symbol is a different cell , and the line is drawn through the mean values. (B) Incomplete inactivation for 300-ms depolarizations to −20 mV. The records are the averaged currents from four depolarizations, in each of five cells. The inset shows the current at the end of the step and the tail current, at a 10× higher amplification. 1 kHz Gaussian filter.
Mentions: The inactivation curve could be described well by a single Boltzmann relation, assuming that channels inactivate fully at depolarized voltages (Fig. 7). The currents recorded during depolarizations do decay to near zero, but small currents are consistently observed at the end of the pulse (Fig. 1 A). This was observed even after depolarizations lasting 120 ms (Fig. 8 A). If the inactivated state is fully absorbing, only 0.0003 of the channels should remain open after 120 ms , but the peak tail current amplitudes correspond to PO,r ∼ 0.02 over a wide voltage range (−60 to +70 mV). The tail currents were small and noisy, so the measured current amplitudes show considerable variability, but residual channel activation was clearly detectable.

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