State-Dependent Inactivation of the α1g T-Type Calcium Channel

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the α1G channel, with symmetrical Na+i and Na+o and 2 mM Ca2+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 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation\*}({\mathrm{e-fold\;for}}\;31\;{\mathrm{mV}};\;{\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation\*}\end{document}, but \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation\*}{\mathrm{{\tau}}}\;=\;12{\raisebox{1mm}{\line(1,0){6}}}17\;{\mathrm{ms\;from}}-40\;{\mathrm{to}}\;+60\;{\mathrm{mV}}\end{equation\*}\end{document}. 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 (∼98%). Inactivation was also produced by long, weak depolarizations \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation\*}({\mathrm{{\tau}}}\;=\;220\;{\mathrm{ms\;at}}\;-80\;{\mathrm{mV}};\;{\mathrm{V}}_{1/2}\;=\;-82\;{\mathrm{mV}})\end{equation\*}\end{document}, which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation\*}({\mathrm{{\tau}}}\;=\;85\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation\*}\end{document}, 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 ≤8% of the channels recovering through the open state. 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 α1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.