Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.
J Membr Biol. 2014 Apr;247(4):319-30. doi: 10.1007/s00232-014-9634-3. Epub 2014 Feb 1.
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 K(V)2.1, 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, confirming 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.
先前的研究表明，在外孔（T449）的一个位点发生点突变，可以使 Shaker 钾通道的慢失活速度加快或减慢约 100 倍。然而，Shaker 中存在两种形式的慢失活这一发现提出了一个问题，即哪种失活过程受突变影响。在仅表现出 U 型失活的 K(V)2.1 通道中，等效突变对失活几乎没有影响，这表明 Shaker T449 的突变仅作用于 C 型失活，这是一个广泛存在但未经检验的假设。本研究重新检查了 Shaker T449 的突变，证实 T449A 加速失活，而 T449Y/V 则使其减慢。T449Y 和 T449V 表现出 U 型失活，这种失活会被高细胞外钾增强，而 T449A 的 C 型失活则会被高钾抑制。对于 12 态 Markov 模型的自动参数估计表明，U 型失活主要发生在弱去极化时的关闭状态，但主要发生在正电压时的开放状态。该模型还表明，WT 通道在本研究中主要表现为 C 型失活，通过关闭失活状态恢复失活，产生电压依赖性恢复。这表明 C 型和 U 型失活都涉及开放失活和关闭失活状态。
