2002 — 2005 |
Fleig, Andrea |
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. |
Magnesium-Nucleotide Depletion Activated Ion Channel
DESCRIPTION (provided by applicant): Metal ions contribute to fundamental biological processes, including maintenance of protein architecture, determination of enzyme catalytic activity and signal transduction. Transport mechanisms for trace metal ions are not well characterized, and no ion channel with the ability to permeate significant amounts of these ions has yet been described. Recently, a superfamily of the putative calcium-permeable cation channel family TRPC (transient receptor potential channels) has been identified. Our data characterize a novel and widely expressed ion channel of this superfamily, LTRPC7, which has an intriguing selectivity profile for trace metal ions. LTRPC7 is regulated by intracellular levels of Mg.ATP and is non-redundantly essential for cell viability. The current proposal characterizes this novel ion channel using electrophysiological and molecular biology techniques. Specific Aim 1 focuses on the biophysical characterization of recombinant and native LTRPC7. First, the channel?s selectivity profile for trace metal ions will be analyzed. Second, since LTRPC7 is dependent on nucleotide triphosphates, these findings will be extended to include other phospho-nucleotides. Third, the involvement of phosphorylation in channel function will be tested. Specific Aim 2 concentrates on the functional and molecular analysis of LTPRC7 and introduces targeted point mutations to evaluate the structural requirements that determine the selectivity and modulation of LTRPC7. Specific Aim 3 addresses the physiological significance of LTRPC7 currents. It is hypothesized that endogenous LTRPC7 contributes to calcium influx pathways normally attributed to the ICRAC conductance in immune cells. Therefore, experiments have been designed addressing the dissociation of these two currents. Finally, the role of enhanced LTRPC7 expression and metal ion entry in pro-apoptotic events and cell death is assessed. The studies proposed above are based on the unique ion channel LTRPC7. By virtue of this channel?s sensitivity to Mg ATP levels and unusual selectivity to divalent ions, LTRPC7 seems to link fundamental processes that adjust plasma membrane divalent cation fluxes according to the metabolic state of the cell. Disturbing this delicate balance may induce LTRPC7-dependent cell death. These features predestine LTRPC7 to be a prominent player in pathophysiological situations mediating the detrimental divalent ion entry in ischaemic events. Detailed understanding of LTRPC7 physiological function will open new and fascinating possibilities to influence cell survival.
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1 |
2007 |
Fleig, Andrea |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 1 Structure and Function of Trpm6/7 Ion Channel Domains @ Univ of Med/Dent Nj-R W Johnson Med Sch |
0.913 |
2007 — 2008 |
Fleig, Andrea |
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. |
Role of Trpv Cation Channels in Mast Cells
DESCRIPTION (provided by applicant): Mast cells are pluripotent effector cells that reside in tissue. They respond to diverse stimuli by releasing potent biological mediators into the surrounding tissue. These mediators include extracellular proteases, histamine and serotonin, as well numerous cytokines, chemokines, and growth factors. Immunologically, mast cells play a crucial role in the generation and maintenance of inflammation, in response to antigenic challenge. In addition to responses to immunological stimulation, mast cells also respond to polybasic secretagogues and physical stimuli. Through responsiveness to physical stimuli (thermal, osmotic and mechanical inputs), mast cells contribute to both tissue homeostasis, and the wounding process that follows noxious insults. Each mechanism for mast cell activation relies on calcium influx through specific plasma membrane cation channels. Here, we report that calcium-permeant cation channels of the TRPV family are expressed in mast cells. TRPV ion channels are a newly recognized family of sensors, which receive, and react to, physical environmental cues, including thermal, osmotic and mechanical stimuli. The central premise of the current proposal is that TRPV channels transduce physiological, and pathophysiological, signals that are functionally coupled to calcium signaling and mediator release in mast cells. We propose to study the regulation, and function, of TRPV cation channels in the mast cell context. Our first Specific Aim tests the hypothesis that expression of TRPV2 confers a specific, thermally-evoked, cation conductance upon mast cells. In the second Specific Aim, we will explore a novel regulatory mechanism for TRPV2. Our preliminary data show that TRPV2 is a target for phosphorylation by protein kinase A in mast cells, and that this interaction is mediated by a novel adapter protein, PAP7. PAP7 specifically bridges TRPV2 and PKA. We will explore the functional consequences and upstream regulatory mechanisms for phosphorylation of TRPV2 by PKA, and the contribution of PAP7. In Specific Aim 3, we will examine the representation and functionality of TRPV channels in dermal mast cells, exploring the hypothesis that TRPVs couple mast cell responses to environmental stimuli in the skin.
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1 |
2008 — 2011 |
Fleig, Andrea |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 1: Structure and Function of Trpm6/7 Ion Channel Domains @ Univ of Med/Dent Nj-R W Johnson Med Sch
Metal ions contribute to fundamental biological processes, such as maintenance of protein structure, enzyme activity and signal transduction. Transport mechanisms for divalent metal ions are not well-characterized. Only recently a novel ion channel, TRPM7, was found to conduct significant amounts of these ions into cells. TRPM7 is a ubiquitously expressed protein combining a channel and a kinase domain (channel kinase). Its channel activity is regulated by intracellular magnesium (Mg2*) and nucleotriphosphates (Mg-NTP). The channel plays a key role in Mg homeostasis due to its unprecedented permeation preference for divalent ions. TRPM7 is highly conserved across species, including zebrafish, emphasizing its critical role in cell physiology. An additional protein, TRPM6, is structurally similar to TRPM7, but mainly expressed in absorptive tissues such as kidney and intestine. Dysfunctional TRPM6 activity leads to hypomagnesemia in humans. We here hypothesize that TRPM7 plays a ubiquitous role in regulating cellular magnesium homeostasis and that co-assembly with TRPM6 enhances its function in specialized absorptive tissues. We hypothesize that the alpha kinase domains of both proteins serve a dual function by regulating ion channel sensitivity to intracelLular Mg2* and Mg-ATP and by phosphorylating substrates unrelated to channel function. Hence, in Aim1 we suggest electrophysiological analyses in collaboration with Project 2 (Ryazanov) and Project 4 (Stokes) investigating the functional consequences of channel kinase heteromerization on magnesium and metal ion influx. This will be pursued in aco-expression system using HEK293 cells and in a native system from cells isolated from wild-type, heterozygous channel kinase knock-out (KO) and kinase-mute mice models. In Aim 2 we will identify structural features of the pore region of TRPM7 and TRPM6 that confer the selectivity filter for divalent ions, including magnesium. In collaboration with Project 2 (Ryazanov) a series of pore mutants will be tested for their permeation profile in both TRPM7 and TRPM6/TRPM7 heteromeric channels, leading to a delineation of the molecular basis for channel selectivity. Together with Project 3 (Scharenberg) we will determine in Aim3 whether the biophysical and functional properties of TRPM7 and TRPM6/TRPM7 are conserved across species. Based on our current knowledge of TRPM7 physiology we will focus on the biophysical characterization of zebrafish channel kinase. Further differential analyses will identify regions of channel kinases responsible for specific physiological functions. Detailed understanding of the functional consequences of TRPM6/TRPM7 interaction and role of their kinase domains will open new possibilities to influence magnesium and metal ion absorption in health and disease.
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0.913 |