title: Slow inactivation in Shaker K channels is delayed by intracellular tetraethylammonium.
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 two-pulse 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 significant 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 significant 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.
authors.affiliation: Centro de Neurociencias de Valparaíso and Departamento de Neurociencias, Universidad de Valparaíso, 2349400 Valparaíso, Chile.
authors.forename: Vivian
authors.initials: V
authors.lastname: González-Pérez
authors.forename: Alan
authors.initials: A
authors.lastname: Neely
authors.forename: Christian
authors.initials: C
authors.lastname: Tapia
authors.forename: Giovanni
authors.initials: G
authors.lastname: González-Gutiérrez
authors.forename: Gustavo
authors.initials: G
authors.lastname: Contreras
authors.forename: Patricio
authors.initials: P
authors.lastname: Orio
authors.forename: Verónica
authors.initials: V
authors.lastname: Lagos
authors.forename: Guillermo
authors.initials: G
authors.lastname: Rojas
authors.forename: Tania
authors.initials: T
authors.lastname: Estévez
authors.forename: Katherine
authors.initials: K
authors.lastname: Stack
authors.forename: David
authors.initials: D
authors.lastname: Naranjo
mesh_terms: D000495:Allosteric Site / Q000187:drug effects / Q000502:physiology; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D004735:Energy Transfer / Q000502:physiology; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology*; D051665:Kv1.4 Potassium Channel / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D051379:Mice; D009865:Oocytes; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000378:metabolism / Q000494:pharmacology; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D019789:Tetraethylammonium / Q000378:metabolism / Q000494:pharmacology*; D013816:Thermodynamics; D014982:Xenopus laevis
chemical_list: D051665:Kv1.4 Potassium Channel; D026902:Potassium Channel Blockers; D019789:Tetraethylammonium; D011188:Potassium