Slow inactivation in Shaker K channels is delayed by intracellular tetraethylammonium

González Pérez Vivian, Alan Neely, Christian Tapia, González Gutiérrez Giovanni, Gustavo Contreras, Patricio Orio, Verónica Lagos, Guillermo Rojas, Tania Estévez, Katherine Stack, David Naranjo

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker ( Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches - the so called C inactivation - is a constriction of the external mouth of the channel pore that prevents K + ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a twopulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was - 90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a signifi cant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814 - 826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a " foot in the door " mechanism, implying a signifi cant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

Original languageEnglish
Pages (from-to)633-650
Number of pages18
JournalJournal of General Physiology
Volume132
Issue number6
DOIs
StatePublished - Dec 2008

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