Multiple regulatory functions of the cyclic nucleotide sensing domain of HCN channels

细胞膜含有受体蛋白，这些蛋白结合天然化合物(信使)，然后通过改变其分子结构来做出反应，以激活特定的生物功能。膜中的其他蛋白质响应电信号(电压)而不是信使进行开关。我们特别关注称为HCN通道的受体，这是一种门控小孔，使离子能够跨膜运输；这种门控既对电压做出反应，也对称为环磷酸腺苷或cAMP的信使的结合做出反应。我的团队最近发现，尽管负责cAMP感测的结构域位于膜外，但这个结构域仍然控制着嵌入膜内的单独的电压感应域。这种控制的结果是响应于电压调制HCN通道孔打开的速度。这在生理上很重要，因为cAMP信使是在兴奋的条件下由身体产生的，比如肾上腺素激增，或者在大脑某些部位癫痫发作时，通道反应的时机至关重要。*值得注意的是，我们发现，响应电压打开通道的速度和相反过程的速度(响应相反的电压关闭)受到相同cAMP感应域控制的不同控制模式的影响。我的团队正在研究这两种不同的控制模式是如何发生的：我们假设两个感应域是接触的，这种接触改变了电压感应域的运动，以响应电信号。我们有三种方法来测试这一点：*(1)我们使用基因工程在我们预测发生结构域接触的位置改变HCN通道的结构，并测试哪种不同的控制模式会因此而改变。*(2)我们将一个荧光分子连接到电压敏感区域，并测量在开关过程中荧光强度是如何变化的。使用这项技术，我们将检验我们的预测，即电压敏感响应直接根据控制模式的存在与否而改变。*(3)我们将化学交联剂应用于通道，并测试两个结构域是否足够接近以连接，这将支持我们的结构域接触假说。*通过这项工作，我们正在了解cAMP敏感结构域如何在膜上的一个单独的结构域上产生多个作用。由于几乎所有的膜受体都有膜外的结构域，这项工作中揭示的原理最终可能有助于合理设计药物，通过与结构域之间重要的接触面结合来控制这些受体。

The membranes of cells contain receptor proteins that bind natural chemical compounds (messengers) and then respond by switching their molecular structure in order to activate a specific biological function. Other proteins in the membrane switch in response to electric signals (voltage) rather than messengers. We are focusing specifically on receptors called HCN channels which are gated pores enabling transport of ions across the membrane; this gating responds both to voltage and to binding of a messenger called cyclic AMP or cAMP. My group recently discovered that although the domain responsible for cAMP-sensing is situated outside the membrane, this domain nonetheless controls the separate voltage-sensing domain embedded within the membrane. The consequence of this control is modulation of the speed at which the HCN channel pore opens in response to voltage. This is physiologically important because the cAMP messenger is generated by the body under conditions of excitation, such as the "fight-or-flight" adrenaline rush, or during epileptic seizures in some parts of the brain, where timing of a channel response is critical.*** Notably, we discovered that the speed of turning the channel ON in response to a voltage, and the speed of the opposite process (turning OFF in response to the opposite voltage) are subject to separate control modes governed by the same cAMP-sensing domain. My group is studying how these two different modes of control can happen: we hypothesize that the two sensing domains are in contact, and this contact modifies the movements of the voltage-sensing domain in response to electrical signals. We have three ways to test this: ***(1) We use genetic engineering to change the HCN channel's structure in the locations where we predict domain contact occurs, and we test which of the different control modes are changed in consequence. ***(2) We attach a fluorescent molecule to the voltage-sensing domain and measure how the fluorescence intensity changes during ON-OFF switching. With this technique we will test our prediction that the voltage-sensing response changes directly according to whether the control modes are present or not.***(3) We apply chemical crosslinking agents to the channels and test whether the two domains are near enough to be linked, which would support our hypothesis of domain contact.*** With this work we are learning how the cAMP-sensing domain can produce multiple actions on a separate domain in the membrane. Since nearly all membrane receptors have domains outside the membrane, the principles uncovered in this work may eventually aid rational design of drugs to control these receptors by binding to functionally important contact surfaces between domains.**