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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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Envelope test for isolation of α1G currents. Depolarizations of variable duration were given to +60 mV (above) or −20 mV (below). The records shown lasted 2, 5, 10, 20, 50, and 100 ms. The peak amplitudes of the tail currents after repolarization for those records (and for steps lasting 0.2, 0.5, and 1 ms) were scaled to match the currents during depolarization, and are shown as open squares superimposed on the records. Cell a8612, 3 kHz Gaussian filtering.
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Figure 2: Envelope test for isolation of α1G currents. Depolarizations of variable duration were given to +60 mV (above) or −20 mV (below). The records shown lasted 2, 5, 10, 20, 50, and 100 ms. The peak amplitudes of the tail currents after repolarization for those records (and for steps lasting 0.2, 0.5, and 1 ms) were scaled to match the currents during depolarization, and are shown as open squares superimposed on the records. Cell a8612, 3 kHz Gaussian filtering.

Mentions: The ionic conditions used in this study were essentially normal (see materials and methods), including 2 mM Ca2+o, except that K+i was replaced by Na+i to minimize currents through any endogenous K+ channels that might be present. HEK 293 cells have occasionally been reported to have endogenous ion channels (Berjukow et al. 1996; Zhu et al. 1998), which could interfere with study of heterologously expressed channels. Especially since the outward currents at positive voltages were unexpectedly large (Fig. 1), we evaluated the presence of contaminating currents using the “envelope” of tail currents produced by depolarizations of different durations. If the recorded currents reflect activity of a single class of channel, the peak amplitude of a tail current must be proportional to the amplitude of the current at the end of the preceding voltage step (Hodgkin and Huxley 1952a). Fig. 2 demonstrates that the tail currents change in parallel with the step current, and that the tail current amplitudes multiplied by a constant scaling factor superimpose on the time course of the current recorded during the step, for steps to −20 or +60 mV. These data indicate that the α1G currents are well isolated in our experimental conditions.


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

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

Envelope test for isolation of α1G currents. Depolarizations of variable duration were given to +60 mV (above) or −20 mV (below). The records shown lasted 2, 5, 10, 20, 50, and 100 ms. The peak amplitudes of the tail currents after repolarization for those records (and for steps lasting 0.2, 0.5, and 1 ms) were scaled to match the currents during depolarization, and are shown as open squares superimposed on the records. Cell a8612, 3 kHz Gaussian filtering.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Envelope test for isolation of α1G currents. Depolarizations of variable duration were given to +60 mV (above) or −20 mV (below). The records shown lasted 2, 5, 10, 20, 50, and 100 ms. The peak amplitudes of the tail currents after repolarization for those records (and for steps lasting 0.2, 0.5, and 1 ms) were scaled to match the currents during depolarization, and are shown as open squares superimposed on the records. Cell a8612, 3 kHz Gaussian filtering.
Mentions: The ionic conditions used in this study were essentially normal (see materials and methods), including 2 mM Ca2+o, except that K+i was replaced by Na+i to minimize currents through any endogenous K+ channels that might be present. HEK 293 cells have occasionally been reported to have endogenous ion channels (Berjukow et al. 1996; Zhu et al. 1998), which could interfere with study of heterologously expressed channels. Especially since the outward currents at positive voltages were unexpectedly large (Fig. 1), we evaluated the presence of contaminating currents using the “envelope” of tail currents produced by depolarizations of different durations. If the recorded currents reflect activity of a single class of channel, the peak amplitude of a tail current must be proportional to the amplitude of the current at the end of the preceding voltage step (Hodgkin and Huxley 1952a). Fig. 2 demonstrates that the tail currents change in parallel with the step current, and that the tail current amplitudes multiplied by a constant scaling factor superimpose on the time course of the current recorded during the step, for steps to −20 or +60 mV. These data indicate that the α1G currents are well isolated in our experimental conditions.

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