1993 |
Rothberg, Brad S |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Effects of Chronic Ethanol On Neurotransmitter Responses |
0.961 |
2004 — 2010 |
Rothberg, Brad S. |
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. |
Structure and Function of Potassium Channels @ Temple Univ of the Commonwealth
BK-type calcium-activated potassium channels serve as cytoplasmic Ca2+ detectors that can rapidly respond to and modulate transmembrane voltage. These channels are critical in controlling action potential firing in neurons as well as smooth muscle contractility, and consequently, dysfunction of BK channels is associated with movement disorders and epilepsy in humans, and with hypertension in mouse models. An understanding of the structural basis for gating in BK channels thus has direct relevance to human disease, and may ultimately lead to novel therapeutic interventions. In the absence of a 3-D structure for the BK channel, we seek to gain structural insights toward mechanisms of Ca2+-dependent channel activation through an understanding of the prokaryotic Ca2+-gated K channels MthK and TvoK, which are amenable to both functional and structural study. BK, MthK, and TvoK all contain RCK domains that immediately follow their pore-lining helices. The common molecular architecture shared by BK, MthK, and TvoK suggests that understanding the conformational dynamics of the prokaryotic relatives will provide mechanistic insight relevant to BK channels. Our approach toward gaining these novel insights will be multidisciplinary, using electrophysiology to assay function and NMR spectroscopy to probe structure. The specific insights we seek to gain concern 1) the relation between Ca2+-dependent movements at the RCK subunit interfaces and channel gating and 2) the [Ca2+]-dependence of individual residue movements in RCK subunits. Our proposed research over the coming two-year period is focused on two aims: 1) To determine the energetic contributions of intersubunit bonds to Ca2+-dependent gating in MthK. We will test the energetic contributions of salt bridges and hydrogen bonds at RCK domain subunit interfaces by targeting the individual sidechain components of these interactions with mutagenesis, and assaying the mutation effects on gating using single-channel electrophysiology. 2) To identify Ca2+-dependent intermediate structural conformations in an RCK domain. Here we will analyze the relation between structural movements and [Ca2+] at the atomic scale using solution NMR spectroscopy, by measuring chemical shift perturbations of individual residues as a function of [Ca2+]. These experiments will enable the resolution of conformational steps in the Ca2+-dependent gating pathway that cannot be resolved using electrophysiological or X-ray experiments.
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1 |
2013 — 2017 |
Rothberg, Brad |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Allosteric Modulation of Ion Channel Domains
INTELLECTUAL MERIT The flow of potassium across cell membranes is controlled by potassium channels (K channels). By regulating the flow of charged potassium ions, K channel proteins play a fundamental role in gating electrical impulses in excitable cells, and play a fundamental role in controling electrolyte balance in virtually all cells. In this project, research will focus on defining the structural basis for activation of a large subfamily of K channels that contain regulator of K-conductance (RCK) domains. RCK domains are modulatory ligand-binding domains that control gating of K channels in a wide range of prokaryotic and eukaryotic organisms by binding calcium ions and other ligands. Despite their fundamental importance, the key molecular interactions that lead to ligand-dependent opening of the channel have yet to be clearly defined. Mechanisms of activation in RCK-containing K channels will be investigated through a combination of electrophysiological and X-ray crystallographic analysis. The approach will involve atomic resolution determination of a series of structures representing different conformations of the RCK domain from the prototypical K channel MthK from the archaebacterium Methanobacterium thermoautotrophicum. Studying the MthK channel has unique advantages over other systems, and will yield high-resolution atomic structural data that can be directly linked to channel function, which will hopefully lead to a profound understanding of basic mechanisms of activation in this ubiquitous class of ion channels.
BROADER IMPACTS This research will use methodologies that straddle the interfaces of biology, chemistry, physics, and mathematics, and the goal is to continue to promote training of beginning investigators in approaches that combine these disciplines. The PI's laboratory has a strong record of training personnel at the undergraduate, post-baccalaureate, graduate, and post-doctoral levels in the fundamentals and theory of ion channel biology and membrane biophysics, as well as the practice of experimental techniques in the laboratory. Past trainees have continued in successful scientific careers in academia, industry, and government agencies. Notably, the majority of the PI's previous trainees have been women and/or minorities, and this project will continue to recruit and train young scientists from underrepresented groups from among outstanding undergraduate participants at Temple University's Undergraduate Research Program (URP), in addition to training of graduate students. The PI also participates in activities that promote science education in local secondary schools in the Philadelphia area, and will broaden these activities in the context of this research proposal.
This project is jointly supported by the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.
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0.915 |
2018 — 2021 |
Rothberg, Brad S. |
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. |
Discovery and Mechanism of Bk Channel Gating Modulators @ Temple Univ of the Commonwealth
PROJECT SUMMARY Large conductance calcium-activated K channels (SLO-1 or BK channels) play a key physiological role in limiting smooth muscle contractility and neuronal excitability. Deletion of BK channel pore-forming (alpha) or modulatory (beta) subunits in gene-targeted animal models can lead to diseases that include arterial hypertension, bladder and erectile dysfunction, and neurological disorders including epilepsy; mutations in human BK channel subunits are linked to generalized epilepsy with paroxysmal dyskinesia (GEPD), asthma, and autism spectrum disorders. BK channel activators could thus become components of treatment regimens for cardiovascular and/or neurological disease. To exploit BK channels as a potential medical target, it will be important to expand our molecular arsenal of BK channel activators and learn their mechanisms of action. Doing so will lead to advances in an overall effort to understand BK channel gating mechanisms and ultimately find new treatments for cardiovascular and neurological disease. Under this proposal, we will achieve these goals through a combination of 1) cell-based fluorescent screening, which is aimed at discovery of novel gating modulators for BK channels comprised of tissue-specific subunit combinations, and 2) systematic electrophysiological experiments to determine whether these drugs modulate BK channel function through interactions with the Ca2+-sensor, voltage-sensor, or pore domains of the channel. This equipment supplement will be used for purchase of an isothermal titration calorimeter (ITC) that is capable of directly determining thermodynamic binding constants in drug-receptor interactions. This tool will greatly enhance our research to yield fundamental insights toward BK channel gating mechanisms that may further lead to new treatments for disease.
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0.928 |