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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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Time course of inactivation and deactivation. (A) Time constants are from single exponential fits, from two protocols. The diamonds are for currents recorded during direct depolarizations (the protocol of Fig. 1), with the fit beginning well after the point of peak inward current, and ending at 60 or 120 ms. The squares are for currents recorded after repolarization from +60 mV (the protocol of Fig. 3), fitted from 0.4–0.6 ms after repolarization to the end of the 40-ms steps. Data are from the same eight cells as Fig. 3 B. (B) Deactivation kinetics at strongly hyperpolarized voltages. Voltage steps were in 15-mV increments, from −90 to −150 mV. The records are from cell a8o29, with 5 kHz Gaussian filtering. The inset shows the time constants, which changed e-fold for 32.8 ± 0.4 mV .
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Figure 5: Time course of inactivation and deactivation. (A) Time constants are from single exponential fits, from two protocols. The diamonds are for currents recorded during direct depolarizations (the protocol of Fig. 1), with the fit beginning well after the point of peak inward current, and ending at 60 or 120 ms. The squares are for currents recorded after repolarization from +60 mV (the protocol of Fig. 3), fitted from 0.4–0.6 ms after repolarization to the end of the 40-ms steps. Data are from the same eight cells as Fig. 3 B. (B) Deactivation kinetics at strongly hyperpolarized voltages. Voltage steps were in 15-mV increments, from −90 to −150 mV. The records are from cell a8o29, with 5 kHz Gaussian filtering. The inset shows the time constants, which changed e-fold for 32.8 ± 0.4 mV .

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)

Time course of inactivation and deactivation. (A) Time constants are from single exponential fits, from two protocols. The diamonds are for currents recorded during direct depolarizations (the protocol of Fig. 1), with the fit beginning well after the point of peak inward current, and ending at 60 or 120 ms. The squares are for currents recorded after repolarization from +60 mV (the protocol of Fig. 3), fitted from 0.4–0.6 ms after repolarization to the end of the 40-ms steps. Data are from the same eight cells as Fig. 3 B. (B) Deactivation kinetics at strongly hyperpolarized voltages. Voltage steps were in 15-mV increments, from −90 to −150 mV. The records are from cell a8o29, with 5 kHz Gaussian filtering. The inset shows the time constants, which changed e-fold for 32.8 ± 0.4 mV .
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Time course of inactivation and deactivation. (A) Time constants are from single exponential fits, from two protocols. The diamonds are for currents recorded during direct depolarizations (the protocol of Fig. 1), with the fit beginning well after the point of peak inward current, and ending at 60 or 120 ms. The squares are for currents recorded after repolarization from +60 mV (the protocol of Fig. 3), fitted from 0.4–0.6 ms after repolarization to the end of the 40-ms steps. Data are from the same eight cells as Fig. 3 B. (B) Deactivation kinetics at strongly hyperpolarized voltages. Voltage steps were in 15-mV increments, from −90 to −150 mV. The records are from cell a8o29, with 5 kHz Gaussian filtering. The inset shows the time constants, which changed e-fold for 32.8 ± 0.4 mV .
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