Inactivation mechanisms in voltage-gated ion channels

离子通道是充满水的，蛋白质内衬的孔，可以为无机离子提供穿过细胞膜的通道，这是肌肉和神经细胞中电信号的基础过程。在电压门控离子通道中，膜电压的变化触发一个称为门控的过程，该过程涉及孔中允许或阻止传导的构象变化。激活门控是指响应于膜去极化而打开通道孔的内口中的门，其允许离子以高达每秒数百万个离子的速率移动通过孔。然而，典型地，通道的这种传导状态是借助于第二孔门（称为失活门）自限制的，其关闭停止传导。因此，失活在去极化反应期间电流的幅度和持续时间的调节中起重要作用。 该提案的重点是允许钾离子（K+）通过的电压门控通道。它被称为KV 4通道，它在决定人类心肌收缩的节奏和强度以及调节大脑神经元之间的化学通讯方面起着至关重要的作用。这些功能作用反映了KV 4通道的一个关键特性：在激活门打开后，孔平均仅导电几十毫秒，然后由于失活而变得不导电。在绝大多数电压门控离子通道中，失活门的关闭在可变延迟之后与通道打开相耦合，即，激活门的打开触发使失活门关闭的构象变化。但KV 4的情况并非如此，当通道关闭时，主要的失活过程发生。待检验的假设是KV 4通道本身没有失活门，而失活代表膜去极化和激活门打开之间的断开或滑动。实验细节提供了两个项目。项目1追求长期已知但迄今被忽视的观察结果，即在KV 4中，药物4-氨基吡啶（4-AP）阻止失活。这是一个潜在的重要线索，因为在其他KV通道类型中，4-AP通过通道孔中心空腔中的结合位点起作用，以使激活门稳定在其闭合构象中。通过在孔中使用点突变要解决的主要问题是KV 4通道中的4-AP结合是否也发生在孔的中心腔中。然后将4-AP在野生型和突变的KV 4通道中的作用的实验衍生数据并入KV 4门控的数值模拟中作为假设的检验。项目2利用了Shaker（原型KV通道）的结构-功能研究结果，这些研究已经确定了与孔的界面和孔内的位点，其突变使激活门稳定在其关闭状态。至于使用4-AP的研究，实验的基本原理是稳定激活门的闭合构象的突变将抑制或阻止从闭合到闭合-失活状态的转变。我们的目标是分析一些KV 4通道突变，看看失活是否受到影响。这项研究有望为KV 4通道生物物理学提供重要的见解，特别是激活门滑动是否是其失活行为的可行解释。无论激活门滑动假说是支持还是拒绝，该研究预计将引起离子通道生物药理学家的广泛兴趣，因为已知电压门控钠和钙通道中会发生闭合状态失活。

Ion channels are water-filled, protein-lined pores that can provide a pathway for inorganic ions across the cell membrane, a process that is fundamental to electrical signaling in muscle and nerve cells. In voltage-gated ion channels, changes of the membrane voltage trigger a process known as gating, which involves conformational changes in the pore that either permit or prevent conduction. Activation gating refers to the opening, in response to membrane depolarization, of a gate in the inner mouth of the channel pore that permits ions to move through the pore at rates as high as millions of ions per second. Typically, however, this conducting state of the channel is self-limited by virtue of a second pore gate, referred to as an inactivation gate, whose closing stops conduction. Inactivation therefore plays an important part in the regulation of the amplitude and duration of current during a depolarizing response. The focus of this proposal is on a voltage-gated channel that allows potassium ions (K+) to pass through it. It is known as the KV4 channel, and it plays a vital role in determining the rhythm and strength of human heart muscle contractions and in modulating chemical communication between neurons in the brain. These functional roles reflect a key property of the KV4 channel: after the activation gate opens the pore conducts, on average, for only a few tens of milliseconds before it becomes non-conductive due to inactivation. In the large majority of voltage-gated ion channels the closing of the inactivation gate is, after a variable delay, coupled to channel opening, i.e., opening of the activation gate triggers a conformation change that enables the inactivation gate to close. But that is not the case in KV4, where the predominant inactivation process occurs when the channel is closed. The hypothesis to be tested is that the KV4 channel has no inactivation gate per se and that inactivation instead represents a disconnection or slippage between membrane depolarization and activation gate opening. Experimental details are provided for two projects. Project 1 pursues the long-known but heretofore overlooked observation that in KV4 the drug 4-aminopyridine (4-AP) prevents inactivation. This is a potentially significant clue because in other KV channel types 4-AP acts, via a binding site in the central cavity of the channel pore, to stabilize the activation gate in its closed conformation. The main question to be addressed by using point mutations in the pore is whether 4-AP binding in KV4 channels also occurs in the central cavity of the pore. Experimentally-derived data of the effects of 4-AP in wild type and mutated KV4 channels will then be incorporated in a numerical simulation of KV4 gating as a test of the hypothesis. Project 2 exploits the results of structure-function studies in Shaker, the prototypical KV channel, which have identified interfaces with the pore and sites within the pore whose mutation stabilizes the activation gate in its closed state. As for the studies with 4-AP, the rationale for the experiments is that mutations that stabilize the closed conformation of the activation gate will inhibit or prevent the transition from the closed to the closed-inactivated state. The goal is to analyze a number of KV4 channel mutations to see if inactivation is compromised. This research is expected to provide important insights into KV4 channel biophysics, and in particular whether activation gate slippage is a viable explanation of its inactivation behaviour. Whether the activation gate slippage hypothesis is supported or rejected, the research is expected to be of wide interest to ion channel biophysicists since closed-state inactivation is known to occur in voltage-gated sodium and calcium channels.