Slow inactivation in Shaker K channels is delayed by intracellular tetraethylammonium.
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.
González-Pérez
Vivian
V
Centro de Neurociencias de Valparaíso and Departamento de Neurociencias, Universidad de Valparaíso, 2349400 Valparaíso, Chile.
Neely
Alan
A
Tapia
Christian
C
González-Gutiérrez
Giovanni
G
Contreras
Gustavo
G
Orio
Patricio
P
Lagos
Verónica
V
Rojas
Guillermo
G
Estévez
Tania
T
Stack
Katherine
K
Naranjo
David
D
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
D051665:Kv1.4 Potassium Channel; D026902:Potassium Channel Blockers; D019789:Tetraethylammonium; D011188:Potassium