cAMP binds to closed, inactivated, and open sea urchin HCN channels in a state-dependent manner
| Autor: | Vinay Kumar Idikuda, Zhuocheng Su, Weihua Gao, Qinglian Liu, Lei Zhou |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Physiology Xenopus Plasma protein binding Gating Membrane Potentials Mice 03 medical and health sciences 0302 clinical medicine Protein Domains biology.animal Cyclic AMP Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels Animals Point Mutation Sea urchin Research Articles Membrane potential biology Chemistry Hyperpolarization (biology) biology.organism_classification Transmembrane domain 030104 developmental biology Sea Urchins Biophysics CAMP binding Ion Channel Gating 030217 neurology & neurosurgery Research Article Protein Binding |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| DOI: | 10.1085/jgp.201812019 |
| Popis: | Mammalian hyperpolarization-activated cyclic-nucleotide–modulated (HCN) channels bind cAMP preferably in the open state. Using sea urchin HCN channels, Idikuda et al. reveal less cAMP binding to the closed state and further reduced binding to the inactivated state and thus demonstrate intricate communication between the gate and ligand-binding domain. Hyperpolarization-activated cyclic-nucleotide–modulated (HCN) channels are nonselective cation channels that regulate electrical activity in the heart and brain. Previous studies of mouse HCN2 (mHCN2) channels have shown that cAMP binds preferentially to and stabilizes these channels in the open state—a simple but elegant implementation of ligand-dependent gating. Distinct from mammalian isoforms, the sea urchin (spHCN) channel exhibits strong voltage-dependent inactivation in the absence of cAMP. Here, using fluorescently labeled cAMP molecules as a marker for cAMP binding, we report that the inactivated spHCN channel displays reduced cAMP binding compared with the closed channel. The reduction in cAMP binding is a voltage-dependent process but proceeds at a much slower rate than the movement of the voltage sensor. A single point mutation in the last transmembrane domain near the channel’s gate, F459L, abolishes inactivation and concurrently reverses the response to hyperpolarizing voltage steps from a decrease to an increase in cAMP binding. ZD7288, an open channel blocker that interacts with a region close to the activation/inactivation gate, dampens the reduction of cAMP binding to inactivated spHCN channels. In addition, compared with closed and “locked” closed channels, increased cAMP binding is observed in channels purposely locked in the open state upon hyperpolarization. Thus, the order of cAMP-binding affinity, measured by the fluorescence signal from labeled cAMP, ranges from high in the open state to intermediate in the closed state to low in the inactivated state. Our work on spHCN channels demonstrates intricate state-dependent communications between the gate and ligand-binding domain and provides new mechanistic insight into channel inactivation/desensitization. |
| Databáze: | OpenAIRE |
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