William N. Zagotta - US grants

The cGMP-activated ion channel of photoreceptors is the fundamental molecular element that generates the electrical response to light in the retina. During phototransduction, the absorption of a photon of light by a rhodopsin molecule results in the reduction of the cytosolic concentration of the second messenger cCMP and the closing of a cGMP- activated channel in the membrane of the photoreceptor outer segment. The closing of this cation selective channel in response to light produces the primary electrical signal that is processed by the visual system. Previous work on the macroscopic light-sensitive current in photoreceptors has indicated that these channels are highly specialized for their role in phototransduction. By cooperatively binding multiple cGMP molecules, these channels behave as rapid molecular switches. The goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations in the cGMP-activated channel. In particular, the proposal focus on studies of the conformational changes in the channel protein that occur during activation by cGMP. The approach will be to study the opening and closing behavior of single cGMP-activated channels and the alterations in this gating behavior produced by site-specific mutations in the channel protein. The channels will be studied using single-channel patch-clamp recording on cGMP-activated channels expressed from the bovine cDNA clone in Xenopus oocytes or mammalian cultured cell lines. Analysis of the single-channel behavior will assess the number of closed and open conformations of the channel, the allowed conformational transitions, and the relative energy of the various conformations. This analysis will be applied to channels that have their structure altered by site-directed mutagenesis. The interactions between subunits of the channel will be examined by analyzing channels containing both normal and mutant subunits. The nature of the functional alterations that result from defined structural changes will provide valuable insights into the molecular mechanisms of the channel function.

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): The fundamental ability of a neuron to fire action potentials in response to synaptic input is largely determined by the electrical properties of its dendrites. One key ion channel that regulates the electrical properties of dendrites is the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel. HCN channels operate at the threshold of excitability so small changes in the number, localization, voltage dependence, and cyclic nucleotide dependence of the channels can have a dramatic impact on the excitability of the cell. Our long term goal is to understand the molecular mechanisms for how HCN channels control neuronal excitability. Recently an auxiliary subunit of HCN channels in neurons was discovered, called TRIP8b. TRIP8b has a profound effect on the trafficking, voltage dependence, and cyclic nucleotide dependence of the HCN channels. In this grant, we propose to study the molecular mechanism for how TRIP8b binds to the HCN channel and regulates channel function. Our approach will be to combine x-ray crystallography, for atomic resolution structural information, with electrophysiology and fluorescence to study the functional channel in its native membrane environment. Our experiments will reveal the structure of the interaction between the HCN2 C-terminal region and TRIP8b at atomic resolution, the stoichiometry of the interaction, and the mechanism for TRIP8b regulation of the cyclic nucleotide-dependent and voltage- dependent gating of HCN2. These findings will provide a significant advance in our understanding of the structure and regulation of HCN channels as they exist in the neuron and further illuminate their role in the physiology and pathology of the brain.

DESCRIPTION (provided by applicant): TRPV1 ion channels are multimodal receptors that can be activated by heat, high [H+]o, voltage, arachidonic acid metabolites, capsaicin (the pungent extract of hot chili peppers), and the signaling lipid PI(4,5)P2 (PIP2). Ca2+/Calmodulin (Ca2+/CaM) and ATP may modulate its activity as well. Our long-term goal is to understand the molecular mechanism by which TRPV1 integrates these multiple physiological stimuli. We and others have previously established that PIP2 directly activates TRPV1. Our recent work indicates that the proximal part of the intracellular C-terminal domain comprises at least part of the PIP2 binding site. However, the inability to control the lipid composition of native membranes, the presence of myriad enzymes and other proteins in cells and excised patches, and the difficulty of specifically labeling intracellular domains of channels within cells have proven serious experimental barriers to understanding regulation of TRPV1 by PIP2 and other activation modalities. We have developed a novel approach to reconstitute purified TRPV1 channels at high density in synthetic Giant Unilamellar Vesicles (GUVs). In this proposal we will apply standard patch-clamp methods, Patch-Clamp Fluorometry (PCF), and Transition Metal Ion FRET (tmFRET) to study purified TRPV1 channels in GUVs of defined lipid composition. Single cysteines engineered into our cysteineless TRPV1 background will be used to site-specifically label channels in the GUVs with fluorophore, completely eliminating the background fluorescence problem. The GUVs used for reconstitution will include synthetic lipids that bind transition metals which act as short- distance FRET quenchers in the novel short-range tmFRET approach we have developed. PCF allows us to simultaneously record the function of the channel with electrophysiology and the rearrangement of the channel with fluorescence. These new tools will allow us to measure dynamics of the intracellular N- and C-terminal domains associated with PIP2 activation as well as with activation by heat, Ca2+/CaM, and ATP.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels were first discovered in photoreceptors where they shape the light response. They exhibit several properties that make them specialized for retinal signaling: 1) they are weakly K+ selective, 2) they are activated by membrane hyperpolarization, instead of depolarization seen in virtually every other voltage-gated channel, and 3) they are regulated by the direct binding of cyclic nucleotides to an intracellular domain. Our long term goal is to understand the molecular mechanisms for these properties to better understand the physiology and pathophysiology of the channels in the brain and heart. In previous funding periods we have made great progress toward achieving this goal. We have solved the molecular structures of HCN and related channels and invented ground- breaking new fluorescence methods that allow us to record molecular rearrangements in intact channels simultaneous with electrophysiological recording. In this funding period, we propose to apply these methods to determine the molecular mechanisms of hyperpolarization activation and cyclic nucleotide modulation. These experiments will lead to the first dynamic picture for how HCN channels regulate the excitability of neurons and cardiomyocytes.

DESCRIPTION (provided by applicant): Ion channels underlie all electrical excitability in the brain and heart, and defects in ion channels are the cause of many human disorders. The KCNH family of channels, ERG, ELK, and EAG, are voltage-dependent potassium channels that are specialized for their role in shaping the electrical activity of neurons and cardiomyocytes. Defects in KCNH channels have been shown to be linked to cognitive defects, increased risk of schizophrenia, cardiac arrhythmias, and cancer. Recently it has become clear that the specialized gating of KCNH channels arises from their unique intracellular domains, the N-terminal eag domain, and the C-terminal C-linker/CNBHD. In this proposal, we will test the hypothesis that the C-linker/CNBHD of KCNH channels is a key regulatory domain that mediates the action of the eag domain and other intracellular regulators. We will build on three exciting new X-ray crystal structures we have solved of the intracellular domains of three different KCNH channels: 1) the C-linker/CNBHD of ELK, 2) the C-linker/CNBHD of ERG, and 3) a complex of the CNBHD and eag domain of EAG. These new structures reveal unexpected features like a direct interaction between the eag domain and the CNBHD, and an intrinsic ligand in the binding site of the CNBHD that regulates gating. They provide a framework to better understand the mechanisms of the gating and regulation of KCNH channels. Toward this goal, we will leverage the power of X-ray crystallography, electrophysiology, and fluorescence to study the structure and rearrangements of the eag domain and C-linker/CNBHD during gating of KCNH channels.

Project summary The goal of this proposal is to determine the molecular mechanisms underlying multi-modal gating of the pain-receptor ion channel TRPV1 by interrogating the structural mechanisms for activation by capsaicin, heat, protons, savory compounds, and signaling lipids. We will use tmFRET and computational methods to determine the conformational change induced by each modality and the coupling between modalities.

Project Summary The goal of this equipment request is to acquire a Fluorescence Lifetime Imaging Microscope (FLIM) upgrade to an existing microscope body/optics. The FLIM upgrade will provide a previously unavailable ability to measure the energetics of activation of pain-transducing TRPV1 ion channels.

Cyclic nucleotide-regulated ion channels are exquisite molecular machines that underlie important physiological functions. Cyclic nucleotide-gated (CNG) channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. The related hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker activity of the heart and many neurons in the brain. These cation selective channels are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to an intracellular domain of the channel. Our goal is to reveal the molecular mechanism for this allostery in CNG channels. Our approach will be to study bacterial CNG channels as a model system for the eukaryotic channels because of the huge advantages they provide for our biochemical methods . We will leverage the power of four different methodologies to determine the structure, conformational heterogeneity, and dynamics of these channels: 1) cryoelectron microscopy (cryo-EM), 2) double electron-electron resonance (DEER), 3) microfluidic rapid freeze quench (µRFQ) in combination with DEER, and 4) Rosetta-based molecular modeling. The proposal includes four investigators who are pioneers in each of these methods. The use of all four methods on the same ion channel under the same conditions is synergistic and ultimately will lead to a comprehensive structural and energetic model for the allostery of this channel. Ultimately a molecular understanding of these channels would inform not only the physiology and pathophysiology of the heart and brain, but also the general mechanisms for allosteric control of many enzymes.