Content area
Full text
J Membrane Biol (2014) 247:319330 DOI 10.1007/s00232-014-9634-3
Shaker IR T449 Mutants Separate C- from U-Type Inactivation
Quentin Jamieson Stephen W. Jones
Received: 4 November 2013 / Accepted: 13 January 2014 / Published online: 1 February 2014 Springer Science+Business Media New York 2014
Abstract Previous studies demonstrated that slow inactivation of the Shaker potassium channel can be made *100-fold faster or slower by point mutations at a site in the outer pore (T449). However, the discovery that two forms of slow inactivation coexist in Shaker raises the question of which inactivation process is affected by mutation. Equivalent mutations in KV2.1, a channel exhibiting only U-type inactivation, have minimal effects on inactivation, suggesting that mutation of Shaker T449 acts on C-type inactivation alone, a widely held yet untested hypothesis. This study reexamines mutations at Shaker T449, conrming that T449A speeds inactivation and T449Y/V slow it. T449Y and T449V exhibit U-type inactivation that is enhanced by high extracellular potassium, in contrast to C-type inactivation in T449A which is inhibited by high potassium. Automated parameter estimation for a 12-state Markov model suggests that U-type inactivation occurs mainly from closed states upon weak depolarization, but primarily from the open state at positive voltages. The model also suggests that WT channels, which in this study exhibit mostly C-type inactivation, recover from inactivation through closed-inactivated states, producing voltage-dependent recovery. This suggests that both C-type and U-type inactivation involve both open-inactivated and closed-inactivated states.
Keywords C-type inactivation U-type inactivation
Voltage-dependent potassium channels Kinetic models
Introduction
N-terminally truncated Shaker channels with inactivation removed (Shaker IR) exhibit a slow inactivation mechanism (Hoshi et al. 1991) which is dramatically affected by mutations at outer-pore residue T449 (Lpez-Barneo et al. 1993). Mutations of T449 can slow or speed inactivation up to 100-fold, and this hot spot of inactivation has been canonically ascribed to affect C-type inactivation. However, since characterization of the T449 mutations, U-type inactivation has been discovered to coexist with C-type inactivation in Shaker IR (Klemic et al. 2001) raising questions as to whether mutations of Shaker T449 affect C-type inactivation, U-type inactivation, or both.
The molecular mechanism underlying C-type inactivation is now well understood and the originally proposed pore collapse mechanism (Ogielska et al. 1995; Liu et al. 1996) has been described in greater molecular...