Hydrogen bonds as molecular timers for slow inactivation in voltage-gated potassium channels.
Hydrogen bonds as molecular timers for slow inactivation in voltage-gated potassium channels.
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DOI：
10.7554/elife.01289
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发表时间：
2013-12-10
期刊：
影响因子：
7.7
通讯作者：
Ahern CA
中科院分区：
文献类型：
--
作者：
Pless SA;Galpin JD;Niciforovic AP;Kurata HT;Ahern CA
电压门控钾（Kv）通道使钾流出和膜复极在兴奋组织。许多KV通道在长时间电压刺激的存在下进行离子电导的逐渐丧失，称为缓慢失活，但调节该过程动力学的原子决定因素仍然不清楚。使用合成氨基酸类似物和串联通道亚基的组合，我们在通道的细胞外表面附近建立了两个H-键，赋予Kv通道进入缓慢失活的机制：Asp 447和Trp 434之间的亚基内H-键和连接Tyr 445和Thr 439的亚基间H-键。任何一种相互作用的破坏都会通过选择性过滤器中的局部破坏触发缓慢失活，而切断Tyr 445-Thr 439 H-键可能会将这种构象变化传递给相邻的亚基。http://dx.doi.org/10.7554/eLife.01289.001蛋白质是由称为氨基酸的小分子的长链组成的。这些链扭曲和弯曲成复杂的三维形状，有时两个或更多的链，或“亚基”，包装成一个蛋白质。这些形状通常是由一些氨基酸之间的氢键结合在一起的。此外，由于蛋白质的形状决定了其功能，因此某些蛋白质必须能够在不同形状之间切换才能正常发挥功能。离子通道是通过细胞膜形成孔的蛋白质，允许离子进出细胞。在神经元和心肌细胞中发现的钾离子通道有四个亚基，它们响应各种信号而打开或关闭中心孔。通道的关闭可以是“快”或“慢”。当通道快速关闭（称为快速失活）时，一小部分蛋白质从细胞内部“堵塞”孔。然而，缓慢失活背后的机制仍然不清楚。它被认为涉及在孔边缘发现的一些大体积氨基酸之间的氢键。然而，通过用不能形成氢键的替代品取代这些氨基酸来验证这一假设是很棘手的，因为20种天然存在的氨基酸中没有一种足够相似，可以作为合适的替代品。现在，Pless等人通过使用合成氨基酸克服了这一限制，这些合成氨基酸形成的氢键比它们所取代的氨基酸形成的氢键更强或更弱。结果表明，两种类型的氢键保持孔开放：一种是在同一亚基中的两个氨基酸之间的键，另一种是在相邻亚基中的氨基酸之间的亚基间键。Pless等人认为，打开通道会引起小的运动，逐渐削弱并最终打破四个亚基之一的这些键。然后，孔内的特定氨基酸自由扭曲，并通过其他三个亚基中类似运动的级联阻塞孔并停止离子流动。因此，这些氢键网络充当预设的断裂点，允许通道关闭，即使是在持续刺激下。由于钾离子通道的活性受到调节，是健康神经元和心肌的基础;了解是什么控制了它们的失活率，可能会导致新的方法来调节它们的活性和治疗重要疾病。DOI：http://dx.doi.org/10.7554/eLife.01289.002网站
Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the kinetics of this process remain obscure. Using a combination of synthetic amino acid analogs and concatenated channel subunits we establish two H-bonds near the extracellular surface of the channel that endow Kv channels with a mechanism to time the entry into slow inactivation: an intra-subunit H-bond between Asp447 and Trp434 and an inter-subunit H-bond connecting Tyr445 to Thr439. Breaking of either interaction triggers slow inactivation by means of a local disruption in the selectivity filter, while severing the Tyr445–Thr439 H-bond is likely to communicate this conformational change to the adjacent subunit(s). DOI: http://dx.doi.org/10.7554/eLife.01289.001 Proteins are made from long chains of smaller molecules, called amino acids. These chains twist and bend into complex three-dimensional shapes, and sometimes two or more chains, or ‘subunits’, are packed into a protein. These shapes are often held together by hydrogen bonds between some of the amino acids. Moreover, since the shape of a protein defines its function, some proteins must be able to switch between different shapes to function properly. Ion channels are proteins that form pores through cell membranes, allowing ions to flow in and out of the cell. Potassium ion channels, which are found in neurons and heart muscle cells, have four subunits that move to open or close the central pore in response to various signals. The closing of the channels can be ‘fast’ or ‘slow’. When the channels are closed quickly (called fast inactivation), a small part of the protein ‘plugs’ the pore from the inside of the cell. However, the mechanism behind slow inactivation remained obscure. It was thought to involve hydrogen bonds between some of the bulky amino acids that are found at the edge the pore. However, testing this hypothesis—by replacing these amino acids with alternatives that cannot form hydrogen bonds—was tricky because none of the 20 naturally occurring amino acids were alike enough to be suitable replacements. Now, Pless et al. have overcome this limitation by using synthetic amino acids that form hydrogen bonds that are stronger or weaker than those formed by the amino acids they are replacing. The results suggest that two types of hydrogen bond keep the pore open: one is a bond between two amino acids in the same subunit, and the other is an inter-subunit bond between amino acids in neighbouring subunits. Pless et al. suggest that opening the channel causes small movements that gradually weaken, and eventually break, these bonds in one of the four subunits. Specific amino acids within the pore are then free to twist and—via a cascade of similar movements in the other three subunits—block the pore and halt the flow of ions. As such, these networks of hydrogen bonds act as pre-set breaking points allowing channels to close, even in response to continued stimulation. Since regulated potassium channel activity underpins healthy neurons and heart muscles; understanding what controls their inactivation rate may lead to new approaches to tune their activity and treatments for important diseases. DOI: http://dx.doi.org/10.7554/eLife.01289.002