Inactivation mechanisms in voltage-gated ion channels

离子通道是充满水的、蛋白质排列的孔隙，可以为无机离子提供穿过细胞膜的途径，这一过程是肌肉和神经细胞电信号的基础。在电压门控离子通道中，膜电压的变化触发了一个称为门控的过程，这涉及到孔中的构象变化，从而允许或阻止传导。激活门控是指响应膜去极化，在通道孔的内口打开一个门，允许离子以高达每秒数百万个离子的速度通过孔。然而，典型地，通道的这种导电状态是自我限制的，由于第二孔门，称为失活门，其关闭停止传导。因此，失活在去极化响应期间电流振幅和持续时间的调节中起着重要作用。这个提议的重点是电压门控通道，允许钾离子（K+）通过它。它被称为KV4通道，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KV4通道的一个关键特性：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，失活门的关闭经过可变延迟后与通道打开耦合，即激活门的打开触发使失活门关闭的构象变化。但在KV4中并非如此，主要的失活过程发生在通道关闭时。待验证的假设是，KV4通道本身没有失活门，失活只是代表膜去极化和激活门打开之间的断开或滑移。提供了两个项目的实验细节。项目1研究的是一个众所周知但一直被忽视的观察结果，即在KV4中，药物4-氨基吡啶（4-AP）可以防止失活。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的结合位点起作用，使激活门稳定在其封闭构象中。在孔隙中使用点突变要解决的主要问题是，KV4通道中的4-AP结合是否也发生在孔隙的中心腔中。4-AP对野生型和突变KV4通道的影响的实验数据将被纳入KV4门控的数值模拟中，作为假设的检验。项目2利用了典型KV通道Shaker的结构-功能研究结果，确定了与孔的界面和孔内的位点，其突变使激活门稳定在关闭状态。对于4-AP的研究，实验的基本原理是稳定激活门闭合构象的突变会抑制或阻止从闭合状态向闭合失活状态的过渡。目标是分析许多KV4通道突变，以查看失活是否受到损害。这项研究有望为KV4通道的生物物理学提供重要的见解，特别是激活门滑移是否是其失活行为的可行解释。无论激活门滑移假说是被支持还是被拒绝，这项研究都有望引起离子通道生物物理学家的广泛兴趣，因为已知在电压门控的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.