1985 — 1986 |
Papazian, Diane M |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Dna Transformation of Drosophila With the Shaker Gene @ University of California San Francisco |
0.972 |
1990 — 1999 |
Papazian, Diane M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Shaker K+ Channels--Function of S4 and Biochemistry @ University of California Los Angeles
The Shaker gene of the fruit fly encodes voltage-dependent K+ channel proteins. The long-term goals of the research are to derive a biochemical model for the structure of the channel, and to correlate its functional properties with its structure. The specific aims are: 1) to study the function of the conserved S4 sequence, which has been proposed to be the voltage-sensor of the channel. Single amino acid mutations will be made in the S4 basic amino acids, acidic residues that might form ion pairs with the S4, and control residues. The mutant channels will be expressed in Xenopus oocytes and analyzed electrophysiologically. 2) to study the disposition of the Shaker proteins in the membrane. The modification of potential sites for posttranslational glycosylation will be studied in in vitro translation reactions and in vivo. If these sites are modified in vivo, their topological location can be inferred. 3) to study the subunit structure of the channel. The Shaker gene will be expressed in a tissue culture system to begin biochemical experiments with the eventual goals of purifying the channel and determining the number of Shaker subunits per channel. K+ channels in the nervous system are implicated in the basic mechanisms of epileptogenesis. K+ channels in smooth muscle are promising pharmacological targets for the control of hypertension and stroke. K+ channels in lymphocytes are altered in a mouse model for lupus erythematosus. Therefore, studying the structure and function of K+ channels will contribute to our understanding of the etiology and treatment of a variety of diseases.
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2000 — 2003 |
Papazian, Diane M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Shaker K+ Channels-Function of S4 and Biochemistry @ University of California Los Angeles
The long term goal of this research is to elucidate the physical mechanism of voltage-dependent activation in K+ channels by identifying structural interactions in the voltage sensor and characterizing their rearrangements during activation. Shaker and ether a go-go (eag) K+ channels will be expressed in Xenopus oocytes for electrophysiological, biochemical, and spectroscopic analysis. Unlike Shaker, eag activation is dramatically modulated by extracellular Mg2+. To obtain unique insights into voltage sensor, in voltage-dependent transitions during activation will be investigated. The specific aims of the proposal are: 1) To test the hypothesis that eag-specific acidic residues in S2 and S3 compose the Mg2+ binding site. 2) To test the hypothesis that the Mg2+ binding site in eag represents a general structural constraint in other K+ channels, including HERG and Shaker. 3) To identify structural constraints in the Shaker voltage sensor. This aim concludes work in the previous period. 4) To test the feasibility of site-directed fluorescent labeling in eag, and then use this approach to test the hypothesis that the S2 segment participates in rate-limiting, Mg2+-sensitive, conformational changes at hyperpolarized potentials during eag activation. Dr. F. Bezanilla of UCLA will collaborate in these experiments. This proposal describes basic research aimed at understanding the structure and function of voltage-dependent ion channels. The research is likely to have significant health relevance because ion channels play essential biological roles in the brain, heart, and skeletal muscle. The research may also contribute to our arrhythmias and neurological seizures. Among K+ channels, eag homologues, which are widely expressed in the brain and heart, are uniquely regulated by Mg2+, and thus may underlie some of these effects.
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2002 — 2005 |
Papazian, Diane M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Quality Control of K+ Channel Biogenesis @ University of California Los Angeles
DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the mechanisms of quality control that ensure that only properly folded and assembled potassium channels are expressed on the cell surface. Potassium channels adopt their native structures in the ER. Channel proteins that fail to fold or assemble properly are recognized by a stringent quality control system and retained in the ER. This system prevents transport of misfolded or incompletely assembled proteins to locations where aberrant functional properties could disrupt cellular physiology. The proposed research focuses on two aspects of potassium channel quality control. First, how are structurally immature, misfolded, or unassembled potassium channel proteins retained in the ER? Second, what pathways dispose of ER-retained potassium channel proteins? What are the roles of proteasomal degradation and aggresome formation, which have been implicated in the disposal of other ER-retained proteins? The proposed research is relevant to the etiology of channelopathies, such as Long QT Syndrome Type 2, in which channel proteins are retained in the ER and may be subjected to ER-associated degradation. We will accomplish the following specific aims: (1) to determine the role of cytoplasmic domains in ER retention and release during biogenesis of Shaker and Kv1.3 potassium channels; (2) to identify mechanisms used by mammalian cells to dispose of ER-retained potassium channel proteins; and (3) to compare the quality control of HERG channel biogenesis in a mammalian cell line and in cardiac ventricular myocytes. Channel proteins will be expressed in HEK293T cells or cultured ventricular myocytes for studies of protein maturation, stability, protease inhibitors. Experimental approaches will include expression and biochemical analysis of wild type and mutant channel proteins, confocal microscopy, cell surface labeling, and electrophysiology. The proposed research will advance our knowledge of basic aspects of ion channel cell biology and improve our understanding of the etiology of channelopathies.
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2004 — 2014 |
Papazian, Diane M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Voltage-Dependent Activation in K+ Channels @ University of California Los Angeles
DESCRIPTION (provided by applicant): In May 2003, the ion channel community was galvanized by the publication of the high resolution X-ray structure of an archaebacterial voltage-dependent K+ channel, KvAP, by MacKinnon and co-workers. KvAP contains sequence hallmarks and functional properties that establish it as a close relative of eukaryotic voltage-dependent K+ channels. The structure of the pore domain in KvAP is similar to KcsA, MthK, and KirBac. In contrast, the structure of the voltage sensor domain in KvAP contains several surprising features that were unanticipated from previous work. Largely influenced by this structure, MacKinnon and co-workers have proposed a new model for voltage-dependent activation in which the voltage sensor domain acts as a hydrophobic paddle that moves through the lipid bilayer during activation. The KvAP structure and the hydrophobic paddle mechanism have generated significant controversy and refocused attention on three essential questions: What is the structure of the voltage sensor? How does it move during activation? How do voltage sensor movements open and close pore gates? Answering these questions is the long-term goal of this research project. The Specific Aims of the proposal are: 1) to investigate the topology of KvAP and proximity between the voltage sensor and pore domains in a native membrane environment; 2) to investigate the mechanism of voltage-dependent activation in KvAP, 3) to investigate voltage sensor/pore domain interactions in Shaker channels, and 4) to investigate voltage sensor conformational changes in ether-[unreadable]-gogo (eag) using combined electrophysiological and optical measurements. This proposal describes basic research aimed at understanding the structure and function of voltage-dependent ion channels. The research is likely to have significant health relevance because ion channels have essential roles in the brain, heart, and skeletal muscle.
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2008 — 2011 |
Papazian, Diane M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Kv3.3 K+ Channels and Neuronal Excitability in Spinocerebellar Ataxia Type 13 @ University of California Los Angeles
DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 13 (SCA13) is a dominant human disease characterized by locomotor problems and substantial volume loss in the cerebellum. SCA13 is caused by mutations in the KCNC3 gene, which encodes the voltage-gated K+ channel, Kv3.3. Two allelic forms of SCA13 have been described. One form emerges in adulthood and is characterized by progressive ataxia and cerebellar degeneration. The other form is evident in infancy and is characterized by severe locomotor problems, mental retardation, and cerebellar malformation. Thus, mutations in Kv3.3 are associated with both developmental and neurodegenerative phenotypes. Due to their specialized gating properties, Kv3 channels confer on neurons the ability to fire action potentials at high frequencies. Kv3 channels also control spike duration and thereby regulate activity- dependent Ca2+ influx. The two allelic forms of SCA13 are caused by different KCNC3 mutations that alter channel activity in distinct ways. The long term goal of this research is to test the hypotheses that SCA13 mutations alter the excitability of cerebellar neurons and do so in different ways, and that these changes in excitability affect the age of onset and lead to the locomotor deficits and changes in cerebellar structure that characterize the disease. The Specific Aims of this proposal are to test the hypotheses that: 1) SCA13 mutations differentially alter the excitability of cerebellar neurons, 2) SCA13 mutations differentially alter cytoplasmic Ca2+ load in response to electrical stimulation and affect neuronal survival in Ca2+- and age- dependent cell death paradigms, and 3) the unique gating properties of Kv3 channels play a previously unsuspected role in neuronal development. These Specific Aims will be accomplished using cerebellar neurons in vitro and a vertebrate model organism, the zebrafish Danio rerio, for electrophysiological, optical, genetic, and behavioral analysis. SCA13 is rare, but analysis of SCA13 disease mechanisms may shed light on the etiology of common neurodegenerative diseases such as Alzheimer's. Given the fact that mutations in K+ and Ca2+ channel genes lead to progressive neuronal cell death in SCA13 and SCA6, it is reasonable to suggest that changes in channel function or expression contribute to susceptibility or etiology in common neurodegenerative diseases. If changes in excitability contribute to neuronal cell death, the possibilities for prevention and treatment of neurodegenerative diseases would be greatly expanded because drugs that target specific channels and modulate excitability exist and continue to be discovered.
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2013 — 2014 |
Papazian, Diane M |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Knock-in Mice With Spinocerebellar Ataxia Type 13 Mutations in Kcnc3 (Kv3.3) Gene @ University of California Los Angeles
DESCRIPTION (provided by applicant): This R03 project will generate two lines of knock-in mice with point mutations in the endogenous Kcnc3 gene, which encodes the Kv3.3 voltage-gated K+ channel. In humans, the analogous KCNC3 mutations cause distinct forms of spinocerebellar ataxia type 13 (SCA13). SCA13 is a rare autosomal dominant disease characterized by substantial atrophy of the cerebellum and locomotor deficits. Depending on the causative mutation, SCA13 presents as an early-onset neurodevelopmental disease or as an adult-onset neurodegenerative disease. The developmental form of SCA13 is evident in infancy or early childhood with intellectual disability, motor delay, persistent motor deficits, and severe cerebellar atrophy. The degenerative form of SCA13 emerges in adulthood with progressive ataxia accompanied by progressive loss of cerebellar volume. The product of the disease gene, Kv3.3, is highly expressed in fast-spiking cerebellar neurons, where it is an essential regulator of excitability. Disease-causing mutations alter Kv3.3 function, suggesting that changes in action potential firing trigger pathogenesis in SCA13. As a monogenic disorder, SCA13 provides an excellent opportunity to investigate the role of excitability in neuronal health and survival, and o identify mechanisms that translate altered excitability into pathogenic changes during development and aging. Animal models are essential to determine the consequences of SCA13 mutations in vivo and to investigate how different mutations in the same gene give rise to distinct clinical phenotypes. To identify mechanisms that contribute to pathogenesis in SCA13, a mammalian model system in which brain development, structure, and function are closely related to humans is crucial. Kcnc3 knock-in mice will provide essential tools for investigating the etiology of SCA13 and for translating the results of basic research into new therapeutic approaches for neurodevelopmental and neurodegenerative diseases. The knock-in mice generated in this project will be the first genetically accurate models of SCA13 in a mammalian model system. They will be made freely available as a resource for the research community. During the two year term of the R03 grant, we propose to accomplish the following Specific Aims: 1) to generate targeting vectors encoding the SCA13 adult- onset R421H or infant-onset F449L mutations in the mouse Kcnc3 gene; 2) to generate two lines of knock-in mice; and 3) to begin to characterize the phenotypes of the knock-in animals.
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