Gary Yellen - US grants

DESCRIPTION: A variety of K channels regulate he action potential in cardiac cells. The HERG channel is unusual in that its primary structure resembles that of many depolarization-activated channels, such as Shaker, whereas functionally it displays properties of the inward rectifier. The inward rectification, however, seems to derive from intrinsic gating properties, rather than the more common blockade mechanism. The proposed research will analyze the gating of exogenously expressed human HERG channels, and the effects of channel blockers. The specific aims are to elucidate the biophysical and molecular mechanisms that account for the voltage-dependent behavior of HERG and to examine the mechanisms of interaction with various pharmacological inhibitors.

[unreadable] DESCRIPTION (provided by applicant): Pacemaking - the generation of spontaneous rhythmic electrical activity in heart and brain cells -- is crucial to life and to normal brain function. "Pacemaker channels," also known as l(f), l(h), or HCN channels, are a specific class of ion channels that underlie this function in many cells. They are members of the voltage- activated ion channel superfamily, but unlike almost all other members of the superfamily, these HCN channels are activated by hyperpolarization. Results from the preceding grant period, and evidence from other labs, support the idea that voltage sensing (movement of a specialized positively-charged protein domain) and gating (opening and closing of the transmembrane pore) in HCN channels are both essentially like that of the other family members. This means that the different behavior of these channels is probably accounted for by a difference in the "coupling" mechanism, whereby movement of the voltage sensor is transduced into opening of the pore. Despite some leads, this is still the most mysterious step in the operation of voltage-gated channels. This proposal will address the coupling question directly, using specific structural constraints -- variable- length bridges between pairs of introduced residues ~ as a tool for learning about the moving parts, their range of motion, and the consequences of constraining this range of motion. In addition to this study of the mechanics of gating and coupling, there will be a quantitative study of coupling energetics in wild-type channels and in a specific set of mutant channels that produce dramatic changes in the gating behavior of the HCN channels. Work from the preceding grant period revealed that some of the "leakage" through wild- type channels that occurs at positive voltages (where the channels should be closed) is due to a voltage- independent mode of channel gating in a subpopulation of HCN channels, which interchanges only slowly with the main voltage-dependent population. Using gating current measurements and tools developed in the last grant period, this project will distinguish this voltage-independent mode from true changes in coupling, which can then be analyzed to learn how natural variants of HCN channels, and mutants, affect the coupling process. The structural constraints and functional studies together will elucidate the physical and energetic mechanism of the key physiological process of coupling between voltage sensors and gates. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to continue a Jointly Sponsored Predoctoral Training Program in Neurosciences that is the major source of support for students in the Ph.D. Program in Neurosciences at Harvard University. The goals of this interdepartmental Ph.D. Program, which was established in 1981, are (1) to organize within a single training faculty the neuroscientists at Harvard Medical School, its affiliated hospitals, and Harvard College; in order to (2) to train research scientists/teachers who have a broad background in neuroscience and who are interested in mental health and diseases of the nervous system to carry out original and rigorous research in important areas of neuroscience. In the first 18 months trainees complete a sequence of core courses ranging from cell and molecular neurobiology to systems neuroscience, as well as collateral courses selected from cell and molecular biology, immunology, statistics, and other subjects appropriate to individual interests. Students rotate through three different laboratories. Full time thesis research follows the course work, laboratory rotations, and qualifying exams. Students are also involved in other training activities including journal clubs, seminars and data presentation. There are currently 76 graduate students in the Program. The total faculty includes 94 members, of whom 61 who are currently most actively involved in graduate education are Training Mentors on the present grant. Considerable effort has gone into making this program a highly interactive group with extensive formal and informal contacts between students and faculty.

DESCRIPTION (provided by applicant): Epilepsy affects roughly 1% of the human population. For the one third of patients who cannot achieve adequate seizure control with existing medications, one effective alternative is dietary treatment with a high fat and very low carbohydrate ketogenic diet (KD). The KD can be remarkably effective, with ~1/3 of patients becoming seizure free, but the strict diet regimen is difficult for patients to comply with. Learning the still mysterious mechanism of the KD would teach us how the brain may naturally protect itself against seizures, and also permit the design of better dietary treatments and better anticonvulsant medications. On the KD, the brain uses circulating ketone bodies (KB's, esp. 2 hydroxybutyrate and acetoacetate) as an alternate to the usual fuel source, glucose. This change in fuel source somehow produces an anticonvulsant action, but the link remains unknown. A good candidate is an ion channel well known for its sensitivity to metabolism the ATP sensitive K+ channel or KATP channel. Experiments on brain slices show that KBs can, on a fairly rapid time scale (10's of minutes) lead to slowing of spontaneous firing in cells of substantia nigra pars reticulata. KATP channels are important for this effect. The demonstration of a short term in vitro effect of ketone bodies on excitability, and the implication of KATP channels in the effect, offer a new avenue for investigating the mechanism of the ketogenic diet. We will follow up on this lead by asking how KATP channels function in two brain circuits important in epilepsy, and to learn more about possible mechanisms by which these channels may become activated with ketone body metabolism. Substantia nigra pars reticulata neurons and hippocampal dentate granule cells will be the main focus of this work. The effects of ketone bodies on KATP channels and other targets, such as gene expression, are likely due to changes in proximal consequences of the metabolic change. Optical probes for reactive oxygen species, for NADH, and for ATP will be used to learn how central neurons respond to excitation in the presence of different fuel molecules. These experiments will report on how metabolism changes during neuronal activation, and how this is affected by KBs, answering fundamental questions about brain metabolism and function. PUBLIC HEALTH RELEVANCE: One of the best treatments for epilepsy (a seizure disorder affecting roughly 1% of the population) is a very low carbohydrate, high fat ketogenic diet. Because the diet is unpalatable and difficult, it would be useful to understand how it acts on brain cells so that better drug therapies (or easier diets) can be designed. This project will study how ketone bodies produced by the body during the diet act on brain cells to change their activity and prevent seizures, by examining electrical activity and metabolic changes in brain slices from rodents.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] We propose to continue a Jointly Sponsored Predoctoral Training Program in Neurosciences that is the major source of support for early year students in the Ph.D. Program in Neurosciences at Harvard University. The goals of this interdepartmental Ph.D. program, which was established in 1981, are (1) to organize within [unreadable] [unreadable] a single training faculty the neuroscientists at Harvard Medical School, its affiliated hospitals, and Harvard College; (2) to train research scientists/teachers who are interested in mental health, diseases of the nervous system, and fundamental mechanisms of the brain. The training program is designed to provide trainees [unreadable] [unreadable] with a broad and thorough background in neuroscience and to mentor them in performing original and rigorous research in important areas of neuroscience. In the first 18 months, trainees complete a sequence of core courses ranging from cell and molecular neurobiology to systems neuroscience, as well as collateral [unreadable] [unreadable] courses selected from cell and molecular biology, immunology, statistics, and other subjects appropriate to individual interests. Students rotate through three different laboratories. Following the coursework, laboratory rotations, and a preliminary examination, students begin full time dissertation research. They are [unreadable] [unreadable] also involved in other ongoing training activities including journal clubs, seminars, and data presentation. There are currently 86 graduate students enrolled in the Program in Neuroscience. The total faculty includes 93 members; the 65 faculty who are currently most actively involved in graduate education are training [unreadable] [unreadable] mentors on this proposal. Considerable effort has gone into making this program a highly interactive group with extensive formal and informal contacts between students and faculty. Graduates of this program have a high rate of staying in careers in biomedical research and make substantial contributions to a growing [unreadable] [unreadable] understanding of neuroscience. [unreadable] [unreadable] [unreadable]

DESCRIPTION Abstract: Metabolic pathways provide essential energy and building blocks for the function of all cells, and dysregulation of these pathways is a central feature of cancer, diabetes, and obesity, which kill or disable millions of Americans every year. The components of core metabolic pathways such as glycolysis have been very well understood for decades, but there are still major gaps in our understanding of their integrated behavior and regulation in the context of living cells. A major challenge to understanding normal metabolism and its dysregulation in human disease is that metabolic behavior can vary dramatically from cell to cell, and over time within a single cell. For example, metabolic state can differ radically between neighboring cell types in a tissue, creating a functional segregation that is important for overall tissue function, or between a single metastatic cancer cell and the surrounding normal cells. Such spatial differences as well as dynamic changes in metabolism within a single cell are invisible to the usual biochemical methods or even modern metabolomic methods, which require disruption of the living cell and homogenization of tissue. Fluorescent sensors of metabolism, engineered by combining fluorescent proteins with metabolite binding proteins, can address this challenge by enabling us to monitor key metabolites in real time, in single living cells, or in hundreds of cells in paralle. We recently piloted the development of novel sensors for two key metabolites (ATP and NADH), in order to address specific neurobiological questions about how metabolism influences neuronal ion channels and can reduce susceptibility to epileptic seizures. But our preliminary results with these sensors have underscored the general problem of cell heterogeneity as well as the need for a much larger toolkit of fluorescent metabolite sensors. We propose to develop a suite of novel sensors for key metabolites in order to address fundamental questions of cellular me

PROJECT SUMMARY/ABSTRACT Genetically-encoded fluorescent biosensors allow us to capture real-time ?movies? of biochemical behavior inside individual cells, and they have already proven to be valuable tools for learning new biology. The most prominent examples are the intracellular calcium sensors, which reveal real-time neuronal activity in the intact brain, but there are many other new tools for measuring glucose concentration, protein kinase activity, caspase action, reactive oxygen species, and core metabolites such as ATP and NADH. Expressed via viral vectors or transgenes, they can be targeted to individual cells or cell types, and thus they can reveal time-dependent changes in signaling or metabolism in these cells in the context of a living, mixed-cell-type tissue ? and virtually all mammalian tissues are composed of multiple cell types with distinct roles in signaling and metabolism. In comparison, biochemical and mass-spec measurements have exquisite chemical sensitivity, but they usually involve sacrificing the preparation (making timecourses hard to learn), and like the also-powerful magnetic resonance spectroscopy/imaging technologies, they rarely have single-cell specificity. But unlike spectroscopic methods, the biosensors must be tailored specifically for each individual target. This generally involves a combination of semi-rational protein engineering ? in which a fluorescent protein and a ligand-binding protein are fused together in a specific way ? followed by screening of random or targeted mutagenic libraries of sensor variants. This screening process is a major limitation for sensor development, and a reason that many published biosensors are not adequately optimized ? meaning that many published sensors are a ?proof of principle? that cannot easily be used, or worse yet, have interferences that make them unreliable reporters of their nominal targets. One reason that optimization is challenging is that many characteristics of a sensor must be simultaneously optimized: the size of the fluorescence response, the sensitivity range for the target, the specificity of the sensor (including interference from other ligands), and resistance to environmental factors such as pH and temperature. We therefore aim to develop a high-throughput and high-content screening approach for genetically- encoded fluorescent biosensors, specifically for those that respond to ligand binding or other chemical stimuli. This screening method uses a series of well-established microfluidic and imaging methods, and we have piloted most of these already. When complete, this screening method should be deployable in other laboratories for widespread use. It will enable the screening of 104-105 sensor variants in less than a day, with information about each sensor variant in a dozen or more different conditions. We will also apply this screening approach to a series of published and unpublished biosensors in need of specific optimizations. This project will enable a dramatic improvement in the availability of high quality biosensors to study new biology.

PROJECT SUMMARY/ABSTRACT Drug-resistant epilepsy is seriously debilitating and very common, affecting about one-third of the 1-2% of people who experience epilepsy during their lifetime. One of the most effective treatments for drug-resistant epilepsy is dietary therapy, in the form of a very-low-carbohydrate, ketogenic diet. Despite its effectiveness, this diet is not very widely used because of the stringency of the diet and the high commitment required of clinicians and other caregivers. It would be very valuable to understand the mechanism by which altered metabolism produces resistance to epileptic seizures, to ?reverse-engineer? it, and to discover alternative pharmacologic ways of tapping into this potent and apparently unique anti-seizure mechanism. We have identified a mouse model that recapitulates the seizure resistance seen in ketogenic diet, but that involves a mutation in a single gene, Bad. The seizure resistance in this genetic model is due to alteration in brain cell metabolism, with less glucose utilization and better utilization of alternative fuels such as ketone bodies, similar to the metabolic changes on a ketogenic diet. We have also discovered a downstream mechanism that is altered both by Bad alteration and by ketogenic diet: a metabolically-sensitive class of ion channels, the ATP-sensitive potassium channels (KATP channels), become more activated in response to metabolic changes. These channels are critical for seizure resistance of the Bad-altered mice, and we have also found that they are responsible for anti-seizure effects of BAD knockout in a brain slice model of seizure. We have also recently learned that KATP channel activation depends on the expression of the BAD protein in individual neurons, which means that the effects of BAD can be genetically targeted to individual cell types or to specific brain regions. This ability to target the genetic manipulation of the BAD protein ? which cannot be done for a global manipulation like diet ? creates the opportunity to learn the cellular sites of action where BAD modification is required to produce seizure resistance. We now have a conditional knockout allele of the Bad gene (Bad flox/flox) that can be used in combination with various ?driver lines? that express Cre recombinase in specific cells. We will determine whether BAD knockout is effective in slice seizure models or against seizures in mice, when the knockout is restricted to certain targets, for instance, to neurons in specific brain regions like the dentate gyrus that are hypothesized to function as ?seizure gates?. We will also test a pharmacological approach to producing the anti-seizure effects of BAD, by asking whether a specific class of BAD-mimetic compounds is capable of reversing or mimicking the effect of BAD knockout on seizure-like events in slices. These studies will advance our mechanistic understanding of metabolic seizure resistance and more generally of endogenous ?seizure gates?, and will explore new pharmacologic approaches to drug-resistant epilepsy.