James S. Trimmer - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

mixed tissue /cell culture

Changes in membrane excitability occur during development, in response to neurotransmitters, hormones and drugs, and in pathological states. These changes are due to differences in not only the functional activity of specific ion channels, but also in the appropriate targeting of receptors and ion channels to specific domains of the surface membrane. This proposal is aimed at determining the fundamental mechanisms that establish and maintain the distribution of voltage-dependent K+ channels in the membranes of excitable cells. We have used a panel of polyclonal and monoclonal antibodies specific for the Kv2.1, and Kv1.5 K+ channel polypeptides in order to determine their distribution in rat central and peripheral neurons, and in the rat pheochromocytoma PC12 cell line. We find that the distinct subcellular distribution of these channels in neurons is recapitulated in the PC12 cell line expressing endogenous Kv2.1 and Kv1.5 channels. In addition, recombinant K+ channel polypeptides expressed in PC12 and MDCK cells from cDNA are sorted properly, indicating that we have established an excellent cell culture system for studying the molecular basis of sorting of these channels. We will used both wild type and transfected PC12 cells, and transfected MDCK cells, to characterize the protein- protein interactions important in determining the subcellular distribution of K+ channels, and the regions of the K+ channel polypeptides mediating these interactions. Together these studies will allow for an understanding of the cellular processes that regulate the distribution of ion channels in the membranes of nerve, heart and muscle cells. The information from these proposed studies complements studies in project 4, which will determine factors governing Na+ and K+ channel distribution in peripheral nerve in primary cultures and in vivo.

Dendritic voltage-gated K+ channels, or Kv channels, are fundamental components of dendritic signalling. Dendritic Kv channels control the characteristics of backpropagating action potentials, and thus influence synaptic efficacy. In addition, these channels can influence the spread of postsynaptic potentials to the soma, influencing the integration of synaptic input and the response of neurons to external stimuli. Lastly dendritic Kv channels can dramatically influence Ca2+ signalling in dendrites, which can have far reaching implications for neuronal plasticity. This proposal is aimed at determining the fundamental mechanisms that determine dendritic function through the regulation of the abundance, distribution and function of dendritic Kv channels. We will focus our studies on the Kv2.1 Kv channel, which underlies a major component of the dendritic delayed rectifier current. This proposal is aimed at determining the dynamic cellular mechanisms that localize Kv2.1 at important sites of Ca2+ signalling in neurons, the phosphorylation sites on Kv2.1 that regulate localization and function, and the role of these Kv2.1-associated neuronal Ca2+ signalling proteins, and other Kv2.1-interacting proteins, in Kv2.1 localization and function. These studies will yield important insights into the reciprocal physiological regulation of Kv2.1 channel activity and Ca2+ signalling in the soma and dendrites of mammalian central neurons. As regulation of dendritic Kv channel activity influences action potential duration, amplitude and frequency, and synaptic efficacy, understanding the mechanisms controlling the composition of Kv channel complexes at the molecular level, anticipated from our proposed studies, will provide insights into the normal and abnormal function of neurons. It will thus contribute to the eventual understanding and treating of a variety of neurological disorders, including diseases associated with altered neuronal excitability such as genetic and acquired epilepsy, cognitive disorders, and affective disorders.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The specific aim of this proposal is to develop a comprehensive library of monoclonal antibodies (mAbs) optimized for use in the brain (i.e. NeuroMabs) at the NINDS/UC Davis NeuroMab Facility. This proposal is driven by the need to greatly expand the availability of such brain-optimized mAbs for use in basic, translational and clinical neuroscience research. Data are being generated at a rapid rate from high throughput post-genomic approaches addressing molecular mechanisms of brain development, neuronal plasticity, and neurological and psychiatric disorders. The validation of candidate genes as potential targets for further basic research, or for the development of therapeutics, relies on characterization of the protein products of these genes. MAbs against defined gone products can serve as the crucial bridge between the inventory of genes expressed in the brain, and insights into how their products determine brain function. However, many of the necessary reagents are either unavailable, or when available suffer from a lack of efficacy and specificity when used in mammalian brain. The availability of high-quality, reliable mAbs that have been optimized for use in human, non-human primate, and rodent brain (i.e. NeuroMabs) is of utmost importance to virtual all areas of neuroscience. The generation of a comprehensive library of NeuroMabs will be pursued by first taking advantage of the wealth of data emerging from the human, mouse and rat genome projects to generate recombinant and/or synthetic immunogens corresponding to fragments of neuronal proteins. These will be used in an intense immunization protocol that yields large numbers of IgG-secreting hybridomas from a relatively short immunization period. These large hybridoma pools will be screened for those mAbs that recognize the cognate antigen in heterologous cells, and then the entire positive pool subjected to comprehensive biochemical and immunohistochemical analyses of their efficacy and specificity in brain. The resultant brain optimized NeuroMabs will be made available at very low cost to the research community as tissue culture supernatants or as concentrated IgG preparations. The NeuroMab secreting hybridomas will also be made freely available. Investigators will use these NeuroMabs for determining the presence and relative abundance of the cognate antigens in developing, adult, aged, and diseased brain, their cellular and subcellular localization, functionally relevant post-translational modifications, and protein-protein interactions. Moreover, NeuroMabs may find additional i applications in direct functional analyses of proteins, in diagnostic procedures, and as therapeutics. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Kv1 channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of Kv1 channels in mammalian hippocampus, specifically in axons and nerve terminals of the perforant path. Using novel, state-of-the-art mass spectrometric approaches we have made great inroads in defining in vivo phosphosites on brain Kv1.2 and Kv22 subunits, and find that many are Pro- associated pSer and conform to consensus binding sites for proteins containing pSer binding modules. Phosphorylation at some of these sites is specific to channels in axons and nerve terminals, and phosphorylation at these sites changes in response to epileptic seizures. Moreover, these sites are likely phosphorylated by proline-directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on Kv1 channel subunits in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native Kv1 channels. In aims 1-2 we will accomplish this by examining the effects of interventions that alter the phospho-state of Kv1.2 and Kv22 subunits. We will mutate identified in vivo ProDK phosphorylation sites, and candidate upstream ProDK priming sites, and intervene in ProDK expression levels in heterologous cells and hippocampal neurons and determine effects on Kv1 channel expression and localization. These experiments will provide insights into the specific role of these in vivo sites in regulating Kv1 channel biology. In aim 3 we will define the effects of epileptic seizures on Kv1.2 and Kv22 phosphorylation, relative to changes in perforant path function in response to acute seizures and during the acquisition of spontaneous recurrent seizures. We will also define the precise cellular and subcellular locations of ProDK-phosphorylated Kv1.2 and Kv22 in normal and epileptic hippocampus. In aim 4 we will identify cellular proteins exhibiting phospho-dependent interaction with Kv1.2 and Kv22 and determine their role in expression and localization. These studies will yield important insights into the physiological and pathological regulation of Kv1 channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for epilepsy.

DESCRIPTION (provided by applicant): Voltage-gated sodium or Nav channels are fundamental components of signaling in mammalian central neurons in that they confer electrical excitability to neurons. Their activity changes in epileptic neurons, and they are prominent targets of anti-epileptic drugs. This proposal is aimed at determining the fundamental mechanisms that regulate the function of the Nav1.2 channel, which plays a crucial role in the excitability of axons and nerve terminals in many mammalian central neurons. Using novel, state-of-the-art mass spectrometric approaches we have discovered an unanticipated complexity of in vivo phosphorylation on Nav1.2 purified from rat brain. These data provide the first opportunity to investigate the role of these novel and unambiguously identified in vivo brain phosphosites on Nav1.2 in governing channel gating. Of particular interest is determining whether these phosphorylation sites regulate the switch between the transient Nav channels that predominate in normal mammalian neurons, and the persistent Nav channels more typical of epileptic neurons. We will also for the first time undertake quantitative proteomic analyses of how in vivo phosphorylation changes in animal models of acute epileptic seizures, and of epileptogenesis in the form of spontaneous recurrent seizures that serve as animal models of human temporal lobe epilepsy. These studies will yield important insights into the physiological and pathological regulation of Nav1.2 channels, which are key regulators of neuronal excitability in mammalian brain and important targets for anti-epileptic drugs. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for epilepsy.

DESCRIPTION (provided by applicant): BK channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of BK channels in mammalian hippocampus. Using novel, state-of- the art mass spectrometric approaches we have made great inroads in defining the in vivo phosphorylation sites on the primary or BK1 subunit of BK channels purified from rat brain. We find that most in vivo phosphorylation sites are Pro-associated pSer and conform to consensus binding sites for proteins containing pSer-binding modules. Moreover, these sites are likely phosphorylated by proline- directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on BK channels in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native BK channels. In aim 1 we will accomplish this by examining the effects of interventions that alter the phosphorylation state of BK1. We will mutate identified in vivo ProDK phosphorylation sites, and will intervene in ProDK expression levels in heterologous cells and hippocampal neurons, and determine effects on BK channel expression. In Aim 2 we will use similar approaches to determine the role of these phosphorylation sites in polarized localization of BK channels. These studies will yield important insights into the physiological and pathological regulation of BK channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for neurological and psychiatric disorders.

DESCRIPTION (provided by applicant): The specific aim of this proposal is to continue the mission of the UC Davis/NIH NeuroMab Facility: to develop a comprehensive library of monoclonal antibodies (mAbs) optimized for use in the brain (i.e. NeuroMabs). This renewal remains driven by the need, articulated in the original proposal to create the UC Davis/NIH NeuroMab Facility, and that still remains, to greatly expand the availability of such brain-optimized mAbs for use in basic, translational and clinical neuroscience research. There remains a need for high- quality antibodies against defined gene products that serve as the crucial bridge between the inventory of genes expressed in the brain, and understanding how their products determine brain function in normal and pathological conditions. However, many necessary reagents remain either unavailable, or when available suffer from a lack of efficacy and specificity, especially when used in mammalian brain preparations. The availability of high-quality, reliable mAbs that have been optimized for use in mammalian brain (i.e. NeuroMabs) is of utmost importance to virtually all areas of neuroscience. We will continue to pursue the generation of a comprehensive library of NeuroMabs by using recombinant and/or synthetic immunogens corresponding to fragments of neuronal proteins in an intense immunization protocol that yields large numbers of IgG-secreting hybridomas from a relatively short immunization period. These large hybridoma pools will be screened for those mAbs that recognize the cognate antigen in heterologous cells, and then the entire positive pool subjected to comprehensive biochemical and immunohistochemical analyses of their efficacy and specificity in brain. The resultant brain-optimized NeuroMabs will continue to be made available at very low cost to the research community as tissue culture supernatants or as purifed IgG preparations. Investigators will continue to use these NeuroMabs for determining the presence and relative abundance of the cognate antigens in developing, adult, aged, and diseased brain, their cellular and subcellular localization, functionally relevant post-translational modifications, and protein-protein interactions. Moreover, NeuroMabs will continue to find additional applications in direct functional analyses of proteins, in diagnostic procedures, and as therapeutics.

See instructions): This is a proposal for the continued training of predoctoral graduate students in the area of molecular and cell biology. The overall guiding principle is to support students from' Training Grant funds who are the most talented and productive. Our objective is to develop their skills as researchers in the general area of cell biology, but we aim to train them more broadly in all aspects of doing research (e.g.giving seminars, writing grants) so that they will go on to be successful in the academy or in research institutions. The program is comprised of 45 Trainer-directed research programs and 15 Trainee positions for graduate students, who will do their dissertation research in the Trainer labs. The graduate students are recruited primarily from four graduate programs (known as Graduate Groups), namely Biochemistry & Molecular Biology, Cell & Developmental Biology, Genetics and Microbiology, and are generally appointed to the Training Grant after their second year in the graduate program. The program is administered by a nine- person Executive Committee, including a student member. The Training faculty will provide research guidance and mentoring in their labs, will be actively involved in recruiting graduate students to the Davis campus, and will offer courses in molecular and cell biology. In the last five years, the Training faculty have placed particular emphasis on the recruitment of under-represented minority students to our program. Currently there is (andwill be) a highly successful training program external seminar series in which extramural speakers are selected and hosted by the Trainees; an Annual Research Retreat, which is held at Lake Tahoe and serves as a forum for presentation of research seminars by students, postdocs and faculty; brown-bag lunches for the Trainees with the Executive Committee to present and discuss their research; a required course in the responsible conduct of research; and quarterly workshops for all current and past trainees in various areas of career development, such as instruction in grant writing. RELEVANCE (See instructions): All of our students will be working in labs where fundamental cellularprocesses are studied at the molecular level, and where the ultimate goal is to understand these processes in the context of human disease. All trainees will take a course in translational research being developed in collaboration with two other training grants on campus in translational research.

DESCRIPTION (provided by applicant): Kv1 channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of Kv1 channels in mammalian hippocampus, specifically in axons and nerve terminals of the perforant path. Using novel, state-of-the-art mass spectrometric approaches we have made great inroads in defining in vivo phosphosites on brain Kv1.2 and Kv22 subunits, and find that many are Pro- associated pSer and conform to consensus binding sites for proteins containing pSer binding modules. Phosphorylation at some of these sites is specific to channels in axons and nerve terminals, and phosphorylation at these sites changes in response to epileptic seizures. Moreover, these sites are likely phosphorylated by proline-directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on Kv1 channel subunits in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native Kv1 channels. In aims 1-2 we will accomplish this by examining the effects of interventions that alter the phospho-state of Kv1.2 and Kv22 subunits. We will mutate identified in vivo ProDK phosphorylation sites, and candidate upstream ProDK priming sites, and intervene in ProDK expression levels in heterologous cells and hippocampal neurons and determine effects on Kv1 channel expression and localization. These experiments will provide insights into the specific role of these in vivo sites in regulating Kv1 channel biology. In aim 3 we will define the effects of epileptic seizures on Kv1.2 and Kv22 phosphorylation, relative to changes in perforant path function in response to acute seizures and during the acquisition of spontaneous recurrent seizures. We will also define the precise cellular and subcellular locations of ProDK-phosphorylated Kv1.2 and Kv22 in normal and epileptic hippocampus. In aim 4 we will identify cellular proteins exhibiting phospho-dependent interaction with Kv1.2 and Kv22 and determine their role in expression and localization. These studies will yield important insights into the physiological and pathological regulation of Kv1 channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for epilepsy.

DESCRIPTION (provided by applicant): The primary goal of this successful predoctoral Training Program in Molecular and Cellular Biology at UC Davis is to provide Program Trainees, who represent the best students in molecular and cellular biology, with the breadth of knowledge and research training that will prepare them for their own successful careers in the national biomedical workforce. Training is provided by 50 Trainers in 14 academic departments, with wide- ranging interests including genetics, biochemistry, structural biology, cell and developmental biology, physiology and neuroscience. Trainers are selected from the top molecular and cellular biologists on campus, each with an active research program and a successful track record of mentoring. Trainees are selected from the top graduate students across six different Graduate groups. The Training Program hones Trainees' critical thinking abilities, oral and written communication skills, career development and networking, and responsible conduct of research. The Training Program also serves as a mechanism for both integrating and appreciating the diverse array of interdisciplinary molecular and cellular biology research performed, and for increasing diversity in graduate education, across the UC Davis campus. This Training Program requests support for 15 predoctoral student slots to support 7-8 trainees for 2 years each, typically during the second and third year of their PhD training, a modest increase given the success that this Training Program has demonstrated. Institutionally, UC Davis has made a major commitment to establish the faculty and infrastructure in molecular and cellular biology to achieve these training goals, and to support such training in the form of this Training Program through substantial institutional support for the future. With such support, this Training Program will make these trainees fluent in the multidisciplinary languages and integrative methods needed to address important problems in molecular and cellular biology, ranging from molecules to disease. While supported by this training grant, Trainees will be involved in 1) research training in Trainers' laboratories and discipline-specific presentations; 2 presentations to interdisciplinary audiences; 3) research skills workshops across all areas of molecular and cellular biology; 4) a career development/networking series; 5) a trainee-organized seminar series; 6) a course on teaching biology to undergraduates; and 7) an annual research retreat, and training in the responsible conduct of research throughout. We believe that this combination of discipline-specific training in Trainers' research laboratories, interdisciplinry scientific training in cutting-edge molecular and cellular biology, and career development offered by this Training Program will make our trainees better prepared for the changing technical and intellectual climate faced by the next generation of basic biomedical scientists.

? DESCRIPTION (provided by applicant): One of the major challenges in neuroscience is to link the structure to the function of neural circuits. To achieve this goal, we need to understand the connectivity between defined neuronal populations and the contribution of these neurons to physiological processes, behavioral responses and disease states. Recent advances in imaging techniques allow us to visualize the brain structure with cellular resolution. Application of the current generation of genetically encoded optical tools, such as sensors and controllers, is facilitating measurement and manipulation of neuron activity from molecular-defined cell populations in awake, behaving animals. However, probing the dynamics of neural circuitry underlying behavior, specifically for dissecting functional-defined circuitry beyond molecular-defined circuitry, not only depends on the improvement of existing tools, but also requires novel engineering. We thus propose to develop a radically novel sensor to label functionally related neurons through biochemical reagents that can integrate neural activity into permanently increased fluorescent signals during a researcher-defined behavioral epoch. Our technology hinges on effector proteins, ion channels, in particular the potassium channel Kv2.1, whose activation status is directly correlated to the integrated neural activity. The activation of Kv2.1is determined by their conformational and post-translational status, and ion channel activation drives electrical signaling. Recently, we have developed molecular tools appropriate for creating probes to monitor the activation of ion channels. Using one-bead-one-compound (OBOC) combinatorial technology, we have identified genetically encoded short peptides (12-16 mers, GESIs) that specifically activate the fluorescence of organic dyes under a given biological condition. Using existing GESIs as scaffolds, we propose to design and screen novel peptide-dye pairs whose interaction is controlled by voltage-induced conformational changes or phosphorylation of Kv2.1, thus transforming the activation status of this abundant neuronal ion channel into fluorescent signals. Our specific aims will start by designing and screening voltage-sensing and dephosphorylation GESIs, guided by our expertise in ion channel structure-function, Rosetta computational protein design and high-throughput OBOC library. We will characterize the expression, cytotoxicity, sensitivity and kinetics of promising Kv2.1- GESI voltage activation and dephosphorylation probes in dissociated neuronal culture and in brain slices. We will finally demonstrate the capability of this novel toolset to identify activated neurons in living animals. A successful outcome of this proposal will enable dynamic mapping of neural activity through a new lens: visualizing the activation states of ion channels that are central effectors of electrical activity in the brain. As this toolset uniquely provides informatio regarding functional connectivity, it represents a completely novel approach for functional circuitry analysis, instead of circuitry dissection based on structure and genetics. Combined with behavior, application of these small dynamic activity tags to brain imaging opens up new dimensions of functional understanding of neuronal circuitry.

? DESCRIPTION (provided by applicant): The availability of high-quality, reliable and monospecific mouse monoclonal antibodies (mAbs) that have been validated and optimized for use in mammalian brain (i.e. NeuroMabs) is of utmost importance to many areas of neuroscience research. Having such reagents for each of the proteins expressed in the brain is necessary to undertake biochemical and immunohistochemical studies to begin to link the nucleic acid sequence information derived from genome-based approaches to the function of the encoded gene products in the brain. Unfortunately, the lack of antibodies against a particular gene product, or antibodies that lack specificity, can severely handicap an entire subfield of neuroscience research and thereby impede progress towards understanding the mechanisms that underlie neurological disease. The UC Davis/NIH NeuroMab Facility works towards this unmet need in two major ways: by generating NeuroMabs for targets that are deemed high-priority by NIH and by distributing them on a low-cost non-profit basis to the neuroscience research community. Seed funds from UC Davis and various NIH sources allowed for the creation of our Facility in 2005 and, over the past nine years, we have undertaken mAb projects against over 400 brain proteins and generated a catalog of over 370 NeuroMabs. Through our contractor Antibodies Incorporated (AI), we have distributed over 44,500 vials of NeuroMabs at low cost to hundreds of researchers at a multitude of different institutions worldwide and our end users have cited the use of NeuroMabs in over 1500 original research publications. Assuming a conservative for-profit price of $350 per 100 µg vial of purified mAb, an estimate of the NeuroMab non-profit savings to end users and their respective funding agencies is over $12.5 million. We have proven ourselves to be an establishment with a strong track record of important contributions and we wish to continue serving neuroscience researchers as best as we can. The worthiness of our Facility to the NIH and its mission has been demonstrated time and again through various funding initiatives over the years, most recently from the NIH Director's Transformative R01 Program. In addition, the combined support and cooperation of the NIH, the Regents of the University of California and AI have enabled us since 2011 to collect program income from the distribution of NeuroMabs to support future Facility self-sufficiency and new mAb research and development. However, the combination of the Transformative R01 funds and the accumulated program income is insufficient to hold onto our experienced senior staff, to keep the Facility running at its present-day level of productivity and to continue doing projects of high priority to NINDS. Maintaining strong support from NIH through additional R24 funding would be instrumental to retaining our valuable human and technological resources, preserving our current UC Davis infrastructure and keeping intact our exceptional protection from royalty and licensing surcharges that would unnecessarily raise the prices of NeuroMabs for our end users.

? DESCRIPTION (provided by applicant): One of the major challenges in neuroscience is to link the structure to the function of neural circuits. To achieve this goal, we need to understand the connectivity between defined neuronal populations and the contribution of these neurons to physiological processes, behavioral responses and disease states. Recent advances in imaging techniques allow us to visualize the brain structure with cellular resolution. Application of the current generation of genetically encoded optical tools, such as sensors and controllers, is facilitating measurement and manipulation of neuron activity from molecular-defined cell populations in awake, behaving animals. However, probing the dynamics of neural circuitry underlying behavior, specifically for dissecting functional-defined circuitry beyond molecular-defined circuitry, not only depends on the improvement of existing tools, but also requires novel engineering. We thus propose to develop a radically novel sensor to label functionally related neurons through biochemical reagents that can integrate neural activity into permanently increased fluorescent signals during a researcher-defined behavioral epoch. Our technology hinges on effector proteins, ion channels, in particular the potassium channel Kv2.1, whose activation status is directly correlated to the integrated neural activity. The activation of Kv2.1is determined by their conformational and post-translational status, and ion channel activation drives electrical signaling. Recently, we have developed molecular tools appropriate for creating probes to monitor the activation of ion channels. Using one-bead-one-compound (OBOC) combinatorial technology, we have identified genetically encoded short peptides (12-16 mers, GESIs) that specifically activate the fluorescence of organic dyes under a given biological condition. Using existing GESIs as scaffolds, we propose to design and screen novel peptide-dye pairs whose interaction is controlled by voltage-induced conformational changes or phosphorylation of Kv2.1, thus transforming the activation status of this abundant neuronal ion channel into fluorescent signals. Our specific aims will start by designing and screening voltage-sensing and dephosphorylation GESIs, guided by our expertise in ion channel structure-function, Rosetta computational protein design and high-throughput OBOC library. We will characterize the expression, cytotoxicity, sensitivity and kinetics of promising Kv2.1- GESI voltage activation and dephosphorylation probes in dissociated neuronal culture and in brain slices. We will finally demonstrate the capability of this novel toolset to identify activated neurons in living animals. A successful outcome of this proposal will enable dynamic mapping of neural activity through a new lens: visualizing the activation states of ion channels that are central effectors of electrical activity in the brain. As this toolset uniquely provides informatio regarding functional connectivity, it represents a completely novel approach for functional circuitry analysis, instead of circuitry dissection based on structure and genetics. Combined with behavior, application of these small dynamic activity tags to brain imaging opens up new dimensions of functional understanding of neuronal circuitry.

Developing enhanced methods for reporting and manipulating brain activity is a major focus of the BRAIN Initiative. A major aspect of these efforts is aimed at developing genetically encoded probes for large-scale sensing and/or manipulation of neural activity in vivo. Major advances have been made in developing probes with enhanced intrinsic properties as to efficacy of reporting and/or manipulating neural activity. However, the utility of these probes in vivo has been limited by an inability to direct their localization to specific subcellular sites in brain neurons. We propose to develop a pipeline of genetically encoded localization modules or GELMs to direct probes for large-scale sensing and/or manipulation of neural activity to specific subcellular sites in brain neurons in vivo. This will yield enhanced signal to noise ratio at any specific site, and allows researchers and clinicians to more effectively report and/or modulate neuronal activity, an important step towards developing new ways to treat, cure, and even prevent brain disorders. An interdisciplinary consortium comprising neurobiologists, binder developers, and neural activity probe developers assembled here proposes development of a very ambitious pipeline for development of a robust and diverse set of genetically encoded localization modules, or GELMs, that when fused to activity reporters and modulators, or ARMs, will lead to the localization and concentration of the fusion proteins at specific subcellular sites in neurons. The ability to effectively and reliably target ARMs, and other genetically encoded probes, to specific subcellular sites in brain neurons in vivo will transform the methodology for large-scale sensing and/or manipulation of neural. The novel high-throughput pipeline for GELM development is based on a convergence of powerful new methods. The first is advances in developing high affinity, specific binders for neuronal target proteins in a format that can be used as intrabodies within neurons. These will be developed into Intrabody-based GELMs or I-GELMs. The pipeline also takes advantage of emerging data on targeting motifs present in otherwise highly related proteins that exhibit highly specific yet distinct subcellular localizations in brain neurons, and that affords an opportunity to develop Targeting motif-based GELMs or T-GELMs based on these motifs. We take advantage of high throughput systems for evaluating the expression, localization and function of GELM-ARM fusions in brain neurons. Lastly, we will make GELMs and GELM-ARM fusions widely available through open source plasmid repositories.

PROJECT SUMMARY This is an application for partial support of the 8th biennial FASEB Science Research Conference on Ion Channel Regulation. The objective of this conference is to stimulate discourse, seed new ideas, and facilitate collaboration in ways that will accelerate new discoveries about ion channels at the basic and translational levels, and to provide a forum for trainees to participate in scientific sessions and in career development activities. Regulation of ion channel function is essential for physiological functions including nervous signaling, pain transduction, and the beating of the heart. Accounting for ~1% of human genes, ion channels are subject to numerous disease-causing mutations. There are more than 55 inherited disorders that are attributed to mutations in genes encoding ion channels. These ?channelopathies? affect the brain (e.g., epilepsy, migraine, ataxia, autism), the heart (e.g., cardiac arrhythmia), and other tissues (e.g., pain, hearing and vision impairment, cystic fibrosis, hypertension, muscle disease). Not surprisingly, ion channels are major drug targets in the treatment of epilepsy, neuropathic pain, hypertension and cardiac arrhythmia. Dysregulation of ion channels is strongly associated with mental illness and cancer, both of which are profound global health concerns. Thus, the topic of our conference is both timely and highly relevant for a broad population of scientists, clinicians, and the general public. The Co-chairs of the 2017 conference will be Drs. Mark Dell?Acqua (University of Colorado Denver) and James Trimmer (University of California Davis); both recognized leaders in ion channel biology with an emphasis on ion channel regulation. The Program consists of nine scientific sessions, one keynote address, and two breakout sessions, one focused on careers in industry, and one on careers in academia/NIH, etc. There will also be career development lunches with individual tables focusing on fellowships, other funding, strategies for applying for postdocs and faculty positions, early career strategies, etc. Most scientific sessions were organized around general themes rather than ion channel subtype, to foster crosstalk between fields typically kept separate in traditional conferences. Several of the planned talks focus upon novel approaches to study ion channel regulation including state of the art techniques in imaging, chemical engineering, generation and characterization of mouse models of human disease, etc. There will be 36 session speakers giving full talks, including at least 17 women (47% of speakers) and 9 session chairs (5 of whom are women, 55%). Most of the invited speakers have not presented at this conference. Of the 36 session speakers, we are planning to include 6 early career stage investigators and 6 members of under-represented minorities in science (17%), numbers that will be bolstered in 14 short talks to be selected from submitted abstracts; we will also work with institutions from historically underfunded states within the West to encourage their participation and promote novel collaborations.

Project Summary Dihydropyridine-sensitive, L-type Cav1.2 and delayed rectifier Kv2.1 channels play critical roles in the regulation of excitability and contraction in arterial smooth muscle. A salient feature of Cav1.2 channels is that they form clusters within which they undergo dynamic, reciprocal interactions that allow functional coupling of adjacent channels and thus amplification of Ca2+ signaling, which is critical to the development of myogenic tone. At present, however, the mechanisms controlling Cav1.2 clustering are unknown. The Trimmer and Santana labs have joined forces to address this fundamental issue. New preliminary data from our labs suggest a novel model that represents a paradigm shift relative to the generally accepted canonical role of Kv2.1, and K+ channels in general, as acting solely as K+ conducting electrical determinants of the intrinsic membrane properties of arterial myocytes. In this model, the Kv2.1 channel has a physical role to increase clustering and thus cooperative gating of Cav1.2 channels. Our data indicate that the balance between the separable electrical and structural roles of Kv2.1 channels fine tunes membrane potential, Cav1.2 clustering, functional coupling of these channels, and hence Ca2+ influx, myogenic tone, and, ultimately, blood pressure. A key finding that underscores the significance of our work is that Kv2.1 expression varies with sex, leading to significant differences in Ca2+ influx and myogenic tone between female and male arterial myocytes. The combination of our complementary skill sets allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the mechanisms controlling Kv2.1 and Cav1.2 organization, and how they impact cell, organ, and whole-body functions under physiological conditions. The project has three specific aims. Aim 1 is to determine the impact of altered Kv2.1 expression levels on clustering and activity of Cav1.2 channels, and myogenic tone in arterial smooth muscle, and on blood pressure. Aim 2 is to define the mechanisms underlying Kv2.1-mediated regulation of Cav1.2 function. Finally, Aim 3 is to use novel genetic models to define the cell autonomous role of Kv2.1, and its separable conducting and non-conducting functions, in regulating Cav1.2 function, and the myogenic response in arterial smooth muscle cells, and systemic blood pressure. The proposed studies have the potential of transforming our understanding of how ion channels are organized in vascular smooth muscle, and provide insights into how arterial diameter and blood pressure are differentially regulated in females versus males.

Sites of contact between the endoplasmic reticulum (ER) and the plasma membrane (PM), termed ER-PM junctions or EPJs, are specialized membrane contact sites present in all cells, and at which physiologically important Ca2+ signaling events, lipid exchange, membrane protein trafficking, and other crucial cell biological processes occur. In many brain neurons, such as hippocampal pyramidal neurons (HPNs) and striatal medium spiny neurons (MSNs), EPJs represent the major Ca2+ signaling microdomain in aspiny regions of the neuron. Neuroproteomic analyses of the macromolecular signaling complexes at dendritic spines has provided information crucial to determining the specific molecular events that underlie normal synaptic signaling, and its dysregulation in neurodevelopmental and adult neurological and psychiatric disorders. A systematic dissection of the macromolecular protein complex present at EPJs at the proteomic level in any cell type, but especially in brain neurons, has not been pursued, due to the lack of appropriate methods and suitable tools. The lack of fundamental information, beginning with a molecular catalog of the protein constituents of these prominent extrasynaptic Ca2+ signaling microdomains, represents a major barrier to our understanding of basic neurophysiology and pathophysiology. We have found that an abundant and broadly expressed neuronal voltage-gated K+ channel, Kv2.1, is specifically localized to large clusters in the PM precisely at sites where EPJs form. Moreover, recent findings show that Kv2.1 actively promotes the formation and/or stabilization of EPJs through direct interaction with a resident ER protein. We propose in this exploratory research proposal to take advantage of the robust and widespread association of Kv2.1 with EPJs to undertake a concerted neuroproteomics effort to identify the protein constituents of this Ca2+ signaling microdomain in HPNs and MSNs. We will immunopurify and/or proximity label protein constituents of Kv2.1-containing EPJs in these neurons, and determine their identify by tandem mass spectrometry. These complementary neuroproteomics analyses will provide a molecular catalog of the protein constituents of these important Ca2+ signaling microdomains in HPNs and MSNs. This information will inform future studies to define the functional role of these constituents in the Ca2+ signaling events that shape the physiology and plasticity of these neurons. Lastly, as dysregulation of protein constituents of EPJs may contribute to the aberrant Ca2+ signaling that leads to degeneration of these important neurons, for example of HPNs in Alzheimer's disease and after stroke, and MSNs in Huntington's disease, they may represent important targets for therapeutic modulation.

Developing enhanced methods for reporting and manipulating brain activity is a major focus of the BRAIN Initiative. A major aspect of these efforts is aimed at developing genetically encoded probes for large-scale sensing and/or manipulation of neural activity in vivo. Major advances have been made in developing probes with enhanced intrinsic properties as to efficacy of reporting and/or manipulating neural activity. However, the utility of these probes in vivo has been limited by an inability to direct their localization to specific subcellular sites in brain neurons. We propose to develop a pipeline of genetically encoded localization modules or GELMs to direct probes for large-scale sensing and/or manipulation of neural activity to specific subcellular sites in brain neurons in vivo. This will yield enhanced signal to noise ratio at any specific site, and allows researchers and clinicians to more effectively report and/or modulate neuronal activity, an important step towards developing new ways to treat, cure, and even prevent brain disorders. An interdisciplinary consortium comprising neurobiologists, binder developers, and neural activity probe developers assembled here proposes development of a very ambitious pipeline for development of a robust and diverse set of genetically encoded localization modules, or GELMs, that when fused to activity reporters and modulators, or ARMs, will lead to the localization and concentration of the fusion proteins at specific subcellular sites in neurons. The ability to effectively and reliably target ARMs, and other genetically encoded probes, to specific subcellular sites in brain neurons in vivo will transform the methodology for large-scale sensing and/or manipulation of neural. The novel high-throughput pipeline for GELM development is based on a convergence of powerful new methods. The first is advances in developing high affinity, specific binders for neuronal target proteins in a format that can be used as intrabodies within neurons. These will be developed into Intrabody-based GELMs or I-GELMs. The pipeline also takes advantage of emerging data on targeting motifs present in otherwise highly related proteins that exhibit highly specific yet distinct subcellular localizations in brain neurons, and that affords an opportunity to develop Targeting motif-based GELMs or T-GELMs based on these motifs. We take advantage of high throughput systems for evaluating the expression, localization and function of GELM-ARM fusions in brain neurons. Lastly, we will make GELMs and GELM-ARM fusions widely available through open source plasmid repositories.

Recombinant Immunolabels for Nanoprecise Brain Mapping Across Scales Understanding brain function and dysfunction requires an understanding of the circuitry of the brain from molecules to cells to circuits. While no single technique can achieve this, a strategic combination of techniques applied across scales can provide information that when integrated can lead to a more complete picture. These techniques can range from analyses of single molecules, including their localization in nanodomains, to subcellular compartments such as synapses and synaptic networks to entire brains. One common theme of these techniques is that they require affinity probes that specifically label subcellular structures, cell types, and circuits within the brain. We propose to enhance an existing resource of renewable affinity probes in the form of an extensive collection of highly-validated monoclonal antibodies. We will enhance our ongoing dissemination of low cost, high quality antibodies to neuroscience researchers by converting these to recombinant form. This will also ensure permanence of this valuable collection of renewable reagents for future researchers. We will use established methods to miniaturize these conventional antibodies into a nanoscale form. These miniaturized antibodies will allow for labeling of brain targets with single nanometer precision, which will provide more spatially precise labeling and overcome the limits to imaging resolution that conventional Abs represent. Moreover, the miniaturized Abs will have much better sample penetration, allowing for more efficient labeling of larger samples of the type used for defining intact circuits. We have formed a consortium of experts who developed and/or are experts in a powerful set of advanced techniques that together allow for brain mapping across scales. This consortium will validate nanoscale antibodies in their respective techniques, a key component of rigor and reproducibility of any antibody-based research. We will also generate and validate novel nanoscale antibodies against targets of great interest to brain investigators for which no suitable reagents exist. Together, these efforts will further enhance the dissemination of this valuable resource, and fundamentally accelerate the pace of brain circuit mapping across scales.

L-type Ca2+ channels (LTCCs) play a fundamental role in brain neurons as mediators of diverse Ca2+ signaling events. LTCCs on neuronal somata play a unique and crucial role in regulating Ca2+-dependent gene expression. A salient feature of LTCCs is that their activity is regulated by clustering through cooperative gating of clustered channels. Their clustering also localizes them to specialized Ca2+ signaling microdomains within which they functionally couple to Ca2+-dependent proteins that transduce the impact of LTCC-mediated Ca2+ entry to specific Ca2+ signaling pathways. Through their canonical function as K+ conducting voltage-gated channels, somatic Kv2.1 channels play critical roles in the regulation of action potentials, with a subsequent impact on LTCC activity. The general consensus is that the functions of LTCCs and Kv2.1 channels in neurons are otherwise largely independent from one another. Our recent work challenges this view. We discovered a novel and unexpected nonconducting role for Kv2.1 in physically regulating the organization of neuronal LTCCs, enhancing their activity and impacting their localization in specific microdomains. These exciting new results lead to a novel model that in brain neurons, Kv2.1 plays dual roles, one as a canonical K+ channel shaping the intrinsic membrane properties of neurons, and the other a nonconducting physical role to cluster LTCCs to enhance their activity and localize them in Ca2+ signaling microdomains. The combination of the complementary backgrounds and skill sets of the Timmer and Santana labs allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the molecular mechanisms whereby Kv2.1 impacts LTCC organization, and the consequences to LTCC function and neuronal signaling. The project has three specific aims, which are to determine how selectively eliminating 1) Kv2.1 expression, 2) Kv2.1 clustering, and 3) the ability of Kv2.1 to enhance LTCC clustering impacts somatic LTCC localization and function, Ca2+-induced Ca2+ release or sparks, and LTCC-dependent transcript factor activation. The proposed studies have the potential of transforming our understanding of how neuronal ion channels are regulated and how this impacts Ca2+ signaling in health and when altered in disease.