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

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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.**