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

细胞膜含有受体蛋白，其结合天然化合物（信使），然后通过转换其分子结构来响应，以激活特定的生物功能。膜开关中的其他蛋白质响应电信号（电压）而不是信使。我们特别关注称为HCN通道的受体，这些通道是门控孔，能够使离子穿过膜;这种门控对电压和称为环AMP或cAMP的信使的结合都有反应。我的团队最近发现，尽管负责cAMP感应的结构域位于膜外，但该结构域仍然控制着嵌入膜内的独立电压感应结构域。这种控制的结果是HCN通道孔响应于电压而打开的速度的调节。这在生理学上很重要，因为cAMP信使是由身体在兴奋条件下产生的，例如“战斗或逃跑”肾上腺素激增，或者在大脑某些部位的癫痫发作期间，其中通道响应的时间是至关重要的。 值得注意的是，我们发现响应于电压而打开通道的速度和相反过程的速度（响应于相反电压而关闭）受到由相同cAMP感测域支配的单独控制模式的影响。我的团队正在研究这两种不同的控制模式是如何发生的：我们假设两个感知域是接触的，这种接触会改变电压感知域对电信号的反应。我们有三种方法来测试这一点： (1)我们使用基因工程来改变HCN通道的结构中的位置，我们预测域接触发生，我们测试的不同的控制模式改变的后果。 (2)我们将荧光分子连接到电压敏感域，并测量荧光强度在ON-OFF切换过程中的变化。利用这种技术，我们将测试我们的预测，电压传感响应直接根据控制模式是否存在而变化。 (3)我们将化学交联剂应用于通道，并测试两个域是否足够接近以连接，这将支持我们的域接触假设。 通过这项工作，我们正在学习cAMP传感域如何在膜中的一个单独的域上产生多种作用。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药物的合理设计，通过结合到结构域之间功能重要的接触表面来控制这些受体。

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.