Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

Gonzalez-Perez, V; Neely A.; Tapia C.; Gonzalez-Gutierrez G.; Contreras, G.; Orio, P; Lagos, V; Rojas G.; Estevez, T.; Stack K.; Naranjo, D


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. © 2008 Palmer and Andersen.

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Título según WOS: Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium
Título según SCOPUS: Slow inactivation in Shaker K channels is delayed by intracellular tetraethylammonium
Volumen: 132
Número: 6
Fecha de publicación: 2008
Página de inicio: 633
Página final: 650
Idioma: English