Ion channels are exquisite molecular machines. By opening and closing an ion selective pore across the cell membrane, these proteins ultimately control everything from our senses to our thoughts. Our long term goal is to understand the precise molecular motions that underlie this gating behavior of ion channels. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our photoreceptors in response to light. They are nonselective cation channels that are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to the channel and modulated by various second messengers. In addition to their role in vision, they are also essential for olfaction and taste, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Their dynamic behavior controls our visual perception, yet the molecular mechanism for their function is largely unknown. This void is due, in part, to a lack of experimental approaches that allow us to """"""""watch"""""""" proteins in action in real time at atomic resolution.
We aim to fill this void by developing novel fluorescence approaches and applying them to investigate the mechanisms of activation and modulation of CNG channels. We will take advantage of two exciting new developments: 1) our solution of the x-ray crystal structures of the intracellular ligand binding and gating domains of the closely related HCN2 and SpIH channels, and 2) our development of methods for simultaneous current and fluorescence measurements from cell-free membrane patches (termed patch-clamp fluorometry, PCF).
Our specific aims are to precisely determine the molecular rearrangement in two important parts of the channel, the cyclic nucleotide- binding domain, and the C-linker, the region that couples binding of cyclic nucleotides to opening of the pore. At the conclusion of these experiments we will know a great deal more about how CNG and related channels work, and will have fully developed new approaches to studying molecular rearrangements applicable to other channels and other proteins.

Public Health Relevance

Ion channels are the transistors of the brain and thereby control everything from our senses to our thoughts. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our rods and cones in response to light, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Our long term goal is to understand how these important proteins work at the molecular level to uncover basic mechanisms of protein function and enable us to develop targeted therapies for neurological diseases.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Neuhold, Lisa
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University of Washington
Schools of Medicine
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Edwards, Thomas H; Stoll, Stefan (2018) Optimal Tikhonov regularization for DEER spectroscopy. J Magn Reson 288:58-68
Gordon, Sharona E; Munari, Mika; Zagotta, William N (2018) Visualizing conformational dynamics of proteins in solution and at the cell membrane. Elife 7:
Dai, Gucan; James, Zachary M; Zagotta, William N (2018) Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels. J Gen Physiol 150:625-635
James, Zachary M; Zagotta, William N (2018) Structural insights into the mechanisms of CNBD channel function. J Gen Physiol 150:225-244
Flynn, Galen E; Zagotta, William N (2018) Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels. Proc Natl Acad Sci U S A 115:E8086-E8095
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James, Zachary M; Borst, Andrew J; Haitin, Yoni et al. (2017) CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel. Proc Natl Acad Sci U S A 114:4430-4435
Dai, Gucan; Zagotta, William N (2017) Molecular mechanism of voltage-dependent potentiation of KCNH potassium channels. Elife 6:
Tait, Claudia E; Stoll, Stefan (2017) ENDOR with band-selective shaped inversion pulses. J Magn Reson 277:36-44
Bankston, John R; DeBerg, Hannah A; Stoll, Stefan et al. (2017) Mechanism for the inhibition of the cAMP dependence of HCN ion channels by the auxiliary subunit TRIP8b. J Biol Chem 292:17794-17803

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