Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state

The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two α subunits are rotated relative to its non-α subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the α subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the αG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the α subunits

Development of a trifunctional non-competitive antagonist suitable for activity-dependent profiling

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.Includes bibliographical references.The muscle-type nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel required for fast synaptic transmission at the neuromuscular junction. It is the archetype of the Cys-Loop superfamily of receptors and a prototypic allosteric protein. The muscle-type AChR has two distinct transmitter binding sites found in the extracellular ligand-binding domain. When acetylcholine binds these sites, a series of still unresolved conformational changes occur, leading to opening of the transmembrane pore over 40 A distant from the binding sites. High resolution structures of the intact receptor and the acetylcholine binding protein have provided greater insight into the structural basis of the allosteric mechanism coupling agonist binding and pore opening. However, comprehensive models of the agonist-bound receptor in its closed and open states are still not available. In particular, the details describing the conformation of binding site residues and the dynamics of their interactions with agonists and competitive antagonists are still under investigation. These details are of particular importance to the design of AChR agonists, partial agonists, and competitive antagonists which may have therapeutic potential for treating neuromuscular and neurological pathologies. Using single-channel electrophysiology we investigated details of the agonist-bound open-state transmitter binding sites. Using a series of structurally related organic cations, we observed a structure-activity relationship that suggests cation-n binding interactions are important for open-state affinity. We also conducted a structure-function study to measure kinetic and thermodynamic differences in agonist binding to the two different transmitter binding sites in both the closed and open states. We observed that the two binding sites have unequal affinities for the agonist choline in the closed state and equal affinities in the open state. The state-dependent difference in affinities suggests that binding determinants from the a subunits predominantly determine open-state choline affinity at each site.(cont.) In the last chapter, we exploit the state-dependent affinities of small molecules for the AChR to develop a probe for live-cell labeling. The ability of a noncompetitive antagonist incorporating state-dependent AChR binding, photoreactivity, and click chemistry moieties was characterized electrophysiologically, and state-dependent photolabeling of AChRs in live cells was demonstrated. A probe with these characteristics is suitable for investigating the activity-dependent changes in AChRs associated with the complex synaptic changes associated with neuromuscular and neurological disorders.by Mathew C. Tantama.Ph.D

Characterizing the roGFP2-Orp1 Fluorescent Biosensor for Detecting Oxidative Stress in Mammalian Cells

Parkinson’s disease is a neurodegenerative disease involving the death of neurons in the substantia nigra and loss of the neurotransmitter, dopamine. The disease leads to progressive loss of motor control. Exact causes and mechanisms by which Parkinson’s disease proceeds are unknown, however, previous experiments determine oxidative stress in mitochondria as a factor that results in cell death. Strategies have been implemented to generate fluorescent biosensors to monitor reactive oxygen species (ROS) concentrations while simultaneously measuring the spatiotemporal distribution and correlation between the ROS, cellular function and organelle. Orp1, an enzyme found in yeast, is a sensitive oxidizing species and when coupled with fluorescent protein, roGFP2, the pair acts as a fluorescent biosensor for the ROS, hydrogen peroxide. In this study, Orp1-roGFP2 protein was expressed and purified from bacterial cell cultures and hydrogen peroxide oxidation assays were conducted to compare performance against characteristics reported in the literature.Orp1-roGFP2 is a fluorescence excitation ratiometric probe and the biosensor signal is obtained by the ratio of fluorescent intensities measured with 390 nm and 480 nm excitation. Sigmoidal kinetics were observed for biosensor oxidation by hydrogen peroxide. We also observed the Orp1-roGFP2 is highly susceptible to air oxidation. Finally the mitochondrial targeting mito-Orp1-roGFP2 gene was subcloned into a GW1 plasmid vector for mammalian expression. Future work will entail transfection of mitochondrially-targeted Orp1-roGFP2 into cultured mouse midbrain neurons to enable live-cell imaging of mitochondrial oxidative stress in cellular models of Parkinson’s disease

Fluorescent Protein Biosensor for Use in Parkinson\u27s Research

Purinergic signaling is a type of extracellular communication that occurs between cells, mediated by adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine. In Parkinson’s Disease, purinergic signaling is disrupted, which contributes to neurodegeneration. In order to monitor this change in cell-to-cell signaling, there is a need for the development of a fluorescent protein (FP) biosensor to study the changes in the concentration of the signaling molecule ATP and its decomposition bioproduct ADP. This summer a genetically encoded ADP sensor that measures changes in ADP concentration was developed. This sensor utilizes Forster Resonance Energy Transfer (FRET) which is a sensing technique that is based on the energy transfer from a donor FP to an acceptor FP. Since this transfer is distance dependent, a change in the sensing domain allows for detection of ADP concentration through changes in fluorescence emission. To develop this FRET based sensor, we are utilizing a cyan-yellow FP pair, as well as a non-fluorescent protein that binds to ADP. Using traditional cloning methods, a small library of ADP sensors from five different versions of both the cyan and yellow proteins was created. This library was screened in E. coli cultures using a method developed to optimize an ATP-sensor. The cloning for this sensor has been confirmed and the library is being tested for sensors responsive to changing concentrations of ADP. With confirmation of a responsive sensor, this sensor design will be validated, allowing for further optimization of this biosensor for the study of purinergic signaling and neurodegeneration

Engineering Bioluminescent Sensors of Cyclic AMP to Study Opioid Signaling

Opioids are small signaling molecules which bind to opioid receptors on the surface of cells. The kappa opioid receptor (KOR) is one of three major types of opioid receptors found in human neurons. When an opioid binds to a KOR, a variety of biochemical signaling pathways are activated inside the cell. Each of these pathways are associated with different physiological effects of KOR activation. The production of a small signaling molecule, cyclic adenosine monophosphate (cAMP), is known to be inhibited during KOR activation of the analgesic (pain-killing) signaling pathway. The ability to interrogate the individual responses of KOR signaling pathways in a living mammal would greatly improve our understanding of how opioids work in the brain. To this end, we have developed a biosensor functioning via bioluminescent resonance energy transfer (BRET) as a tool for both fluorescent and luminescent ratiometric quantification of cAMP. We couple two fluorescent proteins, emitting at different wavelengths, to a luciferase which provides chemiluminescent excitation energy for the complex. The intensity of the two emitted wavelengths vary inversely to each other in response to the presence of cAMP. Calculating the ratio of the two emission intensities creates a metric for cAMP concentration that is normalized to the concentration of our sensor, allowing quantitative comparison across trials. The application of our sensor for dual-color live-cell microscopy was demonstrated in mammalian cells using fluorescence and bioluminescence microscopy. Further proof-of-principle studies in KOR-expressing mammalian cells demonstrates the viability of our sensor for live-cell KOR signaling

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically-encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research

FRET Biosensors: Engineering Fluorescent Proteins as Biological Tools for Studying Parkinson’s Disease

Parkinson’s Disease (PD) is a common neurodegenerative disease with over 200,000 new cases each year. In general, the cause of the disease is unknown, but oxidative stress inside of neurons has been associated with the disease’s pathology for some time. Currently, techniques to study the onset of PD inside of neurons are limited. This makes treatments and causes difficult to discover. One solution to this has been fluorescent protein biosensors. In short, these proteins can be engineered to glow when a certain state is achieved inside a cell. The present research discusses the engineering of a genetically-encoded fluorescent protein (FP) sensor able to detect reactive oxygen species (peroxide, hydroxyl, superoxide, etc.) inside of neurons, giving one the ability to enhance their understanding of the role these species play in the onset of the disease. This sensor relies on Förster Resonance Energy Transfer (FRET) between a green fluorescent protein and a red fluorescent protein to facilitate red-shifting of the sensor’s emission spectrum. Linked via a short polypeptide chain, the energy transfer efficiency of these combined FPs can vary greatly. Various linker lengths and FP combinations were experimentally tested to draw conclusions about their performance. The current trajectory of the research currently implies that those combinations with the shortest linker lengths will yield the highest-performing sensors. This sensor is another vital piece in the library of tools which can be used to help us begin answering the many questions we have about PD and its pathology

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems

pH- and Temperature-Dependent Peptide Binding to the Lactococcus lactis Oligopeptide-Binding Protein A Measured with a Fluorescence Anisotropy Assay

Bacterial ATP-binding cassette transporters are a superfamily of transport systems involved in the import of various molecules including amino acids, ions, sugars, and peptides. In the lactic acid bacteria Lactococcus lactis, the oligopeptide-binding protein A (OppA) binds peptides for import to support nitrogen metabolism and cell growth. The OppA protein is of great interest because it can bind peptides over a broad variety of lengths and sequences; however, current methods to study peptide binding have employed low throughput, endpoint, or low dynamic range techniques. Therefore, in this study, we developed a fluorescence anisotropy-based peptide-binding assay that can be readily employed to quantify OppA function. To test the utility of our assay, we characterized the pH dependence of oligopeptide binding because L. lactis is commonly used in fermentation and often must survive in low pH environments caused by lactic acid export. We determined that OppA affinity increases as pH or temperature decreases, and circular dichroism spectroscopy further indicated that acidic conditions increase the thermal stability of the protein, increasing the unfolding transition temperature by 10 °C from pH 8 to pH 6. Thus, our fluorescence anisotropy assay provides an easy technique to measure peptide binding, and it can be used to understand molecular aspects of OppA function under stress conditions experienced during fermentation and other biotechnology applications

p19(ARF) Is Dispensable for Oncogenic Stress-Induced p53-Mediated Apoptosis and Tumor Suppression In Vivo

Recent studies have shown the p19(ARF) tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that E2F1, a transcription factor known to induce p19(ARF) expression, was required for the response. Here, we use a genetic approach to test whether p19(ARF) functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in p19(ARF) has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21 (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19(ARF) gene. Thus, although p19(ARF) is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli