1975 — 1979 |
Karlin, Arthur |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mechanism of Permeability Control by the Acetylcholine Receptor |
1 |
1979 — 1981 |
Karlin, Arthur |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mechanism of Permeability Control by Acetylcholine Receptors |
1 |
1985 — 1996 |
Karlin, Arthur |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Membrane Receptors and Transport Proteins @ Columbia Univ New York Morningside |
1 |
1985 — 2003 |
Karlin, Arthur |
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. |
Structures of Acetylcholine Receptors @ Columbia Univ New York Morningside
The acetylcholine (ACH) receptor of electric tissue and skeletal muscle transduces the binding of ACH into a change in the permeability fo the post-synaptic membrane to cations. The receptor has the polypeptide chain composition alpha2/Beta/gamma/delta. The overall objectives of the project are to determine the three dimensional structure of the receptor in the membrane and the location within this structure of the functional sites, such as the ACH binding sites and the cation- conducting channel. We will identify the amino acid residues contributing to the ACH binding sites and to the noncompetitive inhibitor (NCI) sites. We will crosslink a Cys 192 and Cys 193, which we have shown are in the ACH binding site, to other residues in the site. We will also directly affinity label other residues in this site. We will probe the NCI sites using a rapid- mixing, rapid-photolabeling technique, which allows us to label the receptor in its transient channel-open and channel-closed states. With this techniques, we will probe the mechanism of block by local anestheties and other NCIs and will test the hypothesis that the NCI sites are within the channel. The amino acid residues that react with labels directed either to the ACH sites or to the NCI sites will be located in the sequences of the chains. We will label susceptible residues in membrane-bound receptor with membrane-impermeant reagents. We will locate these residues in the sequences of the chains in order to determine the folding pattern of the chains in the membrane. We propose to determine the three dimensional structure of the receptor by x-ray crystallography. The location of the functional sites of the receptor in a high resolution structure will provide a basis for our understanding of the function of the receptor in molecular terms.
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1 |
1994 — 1998 |
Karlin, Arthur |
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 of Acetylcholine Receptors @ Columbia University Health Sciences
The nicotinic acetylcholine (ACh) receptors convert the binding of ACh into the opening of a cation-specific channel. The long-term goal of this project is to understand the function of the receptors in terms of their molecular structure. The overall function can be separated into three sub-functions: ACh binding, channel opening and closing, and cation-conduction. Each sub-function is associated with a type of site: the ACh binding sites, the gate and the channel. The proposed research aims at identifying, and locating in the receptor structure, the amino acid residues that form each of these sites. The channel is formed by mostly hydrophobic, membrane-spanning segments of the five receptor subunits (alpha2betagammadelta). The only residues in these segments that are accessible to water and to ions are those that line the channel lumen. To identify the channel-lining residues, most of the residues in the membrane-spanning segments of the alpha subunit will be mutated, one at a time, to cysteine. The mutant alpha, together with wild-type beta, gamma, and delta, will be expressed in heterologous cells. Mutants with near-normal function will be probed with charged, highly-water soluble, lipid-insoluble reagents which are small enough to enter the channel and which react rapidly and specifically with sulfhydryls. The reaction of these reagents with engineered cysteines in the channel will be detected electrophysiologically as an irreversible block of the ACh-induced conduction. In the cation-specific channel, positively charged reagents will react much faster than negatively charged reagents. The pattern of exposure of consecutive residues will indicate their secondary structure. The distance between pairs of engineered cysteines and their mutual exposure in the channel lumen will be probed with a positively charged bifunctional reagent. The gate will be located by probing the closed channel from both ends. The residues lining the ACh binding sites will be identified by a similar approach. Starting with the few residues already associated with these sites, individual consecutive residues will be mutated to cysteine. Those that line the water-accessible surfaces of the ACh binding sites will react with the reagents, more rapidly with the positively charged ones. Reactions at the binding site will block ACh binding and be retarded in the presence of ACh. Pairs of engineered cysteines will be crosslinkable across the binding site. Thus, a map of these sites can be achieved.
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1 |
1999 |
Karlin, Arthur |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Mechanism of Nicotimic Acetylcholine (Ach) Receptor @ Michigan State University
The nicotinic acetylcholine (ACh) receptors convert the binding of ACh into the opening of a cation-specific channel. The long-term goal of this project is to understand the function of these receptors in terms of their molecular structures. The channel is lined by the first two membrane-spanning segments (M1 and M2) of each of the five subunits. The location of the gate opened by ACh, relative to the residues in M1 and M2 and in the cytoplasmic loop between M1 and M2, will be determined in both ? and ?. These residues will be substituted by cysteine (Cys) and the mutants will be expressed in cultured cells. The accessibility of these engineered Cys to a small, positively charged, sulfhydryl-specific reagent from the extracellular side and from the intracellular side will determined both in the closed state and in the open state. The cells will be patch-clamped, and the reaction of the reagent will be detected by the irreversible change in ACh-induced current. The principle to be used is that the accessibility of a residue on the opposite side of the gate from the reagent is affected more by the opening of the gate than the reaction of a residue on the same side of the gate as the reagent. The question of whether the channel is closed by different gates in the resting state and in the desensitized state and how the structure of the channel in the desensitized state compares to the structures in the resting and open states (previously probed) will be approached also by determining the reaction rates of the probe reagent with engineered Cys in the desensitized channel. The dimensions of the ACh binding site formed in the interface between the ? and ? subunit will be estimated from the susceptibility of pairs of Cys, one in each subunit, to crosslinking by bifunctional reagents of different lengths. The Cys will be substituted for residues known to contribute to the binding of ACh.
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0.954 |
2007 — 2011 |
Karlin, Arthur |
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. |
Administrative Core @ Columbia University Health Sciences |
1 |
2007 — 2011 |
Karlin, Arthur |
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. |
Allosteric Modulation of Cardiovascular Ion Channels @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): This Program Project Grant (PPG) proposal comprises 4 Projects and 3 Cores. The focus of the projects is to gain a better understanding of mechanisms modulating cardiovascular ion channels via allosteric effects. A central hypothesis is that allosteric modulators of ion channels play key roles in regulating cardiovascular physiology and are important novel therapeutic targets for major forms of heart disease including heart failure, arrhythmias and hypertension. We propose that perturbation of ion channel function, primarily by means of allosteric effects of subunit interactions and the actions of small molecules on the channel complexes, can regulate ion channels including K+ channels and the ryanodine receptor/Ca2+ release channel in the sarcoplasmic reticulum. We further propose that these allosteric modulations are key novel therapeutic targets that may alleviate many of the undesirable side effects of ion channel pore blockers. Therefore, the goal of this project is to use diverse approaches including biochemical, biophysical, chemical and in vivo testing to address the potential importance of allosteric modulation of cardiovascular ion channels in normal and pathologic functions. The 4 projects are integrally linked: 1) Overall and Project 1 PI - Arthur Karlin has 3 aims focused on identifying sites of interaction for allosteric modulations of ryanodine receptors and BKCa channels. 2) Project 2, PI - Andrew R. Marks has 3 aims focused on the functional characterization of the effects of allosteric modulation of the cardiac and skeletal ryanodine receptors. 3) Project 3 PI - Robert Kass has 3 aims focused on characterization of the allosteric modulation of KCNQ1/KCNE1. 4) Project 4 - PI Steven Marx has 3 aims focused on allosteric modulation of BKCa. Three Cores are: A) Administrative (A. Karlin and A.R. Marks); B) Chemical synthesis core- design and synthesis small molecules that will be used by all 4 projects. C) Animal Models and Tissue Culture Core- will generate and provide cell culture and genetic animal models of ion channel diseases to each project. As noted above all 4 projects are tightly linked. Animal models will be used by Projects 2, 3, and 4 to examine the effects on ion channel function and will be made available to outside investigators. Our group has generated substantial preliminary data to support all of the proposed aims and several publications, including several that are co-authored by members of the group.
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1 |
2007 — 2011 |
Karlin, Arthur |
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. |
Allosteric Site Structures of Cardiovascular Channels @ Columbia University Health Sciences
The cardiac ryanodine receptor calcium-release channel (RyR2) and the smooth-muscle large-conductance calcium-and-voltage-activated potassium (BK) channel are vital participants in normal and pathological cardiovascular physiology. Each is a tetramer of pore-forming subunits in a complex with auxiliary subunits that allosterically modulate channel behavior. FK506-binding protein (FKBP12.6) modulates RyR2, and betal modulates BK channel alpha subu'nit. In addition, these channels are allosterically modulated by ligands with therapeutic potential, such as RyR2 by JTV519. To understand the mechanisms of these allosteric interactions, the amino acid residues in the binding sites for FKBP12.6 and for JTV519 on RyR2 and for betal subunit on BK channel alpha subunit will be identified. Furthermore, changes in the contacts in different functional states of the channels will be determined. Binding-site residues in RyR2 in cardiac sarcoplasmic reticulum membrane, and in RyR2 and BK channel heterologously expressed in cultured cells, will be tagged by photoaffinity labeling, chemical crbsslinking, and foot-printing methods. The residues will be identified by protein cleavage, isolation of labeled peptides, mass spectrometry and micro-sequencing. In addition, cross-linking reactions will be directed to cysteine residues substituted by site-directed mutagenesis at specific locations on the target proteins, and cross-linking will be detected by Western blotting. Modifications of known methods are proposed to make feasible the detailed characterization of binding sites on a low abundance channel like BK and on a very large protein like the RyR2 subunit. The results will provide insights into the molecular mechanisms of allosteric interactions in these two channels and will provide molecular structural bases for cardiovascular therapeutics targeted at these channels.
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1 |
2007 — 2014 |
Karlin, Arthur |
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. |
Bk Channel Modulation by Beta Subunits @ Columbia University Health Sciences
BK channels are large -conductance, voltage-and-Ca-activated K channels (maxi-K, slo), consisting of a tetramer of alpha subunits and up to four beta subunits. Beta"!, one of four types of beta subunits, is found in smooth muscle and modulates the voltage and Ca sensitivities and the kinetics of activation and deactivation of BK channels. The overall goal of this proposal is the determination of the structural basis for the modulation of alpha by betal, about which little is known. It is proposed to map their physical interactions in both the closed state and open state of the channel and to determine the functional consequences of these interactions. The approach is to mutate to cysteine (Cys) four consecutive residues, one at a time, in the extracellular flanking region of each transmembrane segment (TM) in alpha and in beta. There are 7 TMs (SO-S6) in alpha and 2 TMs (TM1 and TM2) in betal. All mutants will be expressed in HEK-293 cells and screened for expression and function. All pairs of functional mutants will be probed in intact cells with a novel membrane-impermeant crosslinker (0.5 -1 nm span) and an oxidizing agent (0.3 nm span), both specific for crosslinking Cys. The extents of crosslinking of BK channel on the cell surface of intact cells with varying crosslinker concentrations and reaction times will be determined by quantitative Western blotting. Relative rate constants of each pair of an alpha Cys and a betal Cys will be calculated and will reflect the proximity of the pair. The functional effects of crosslinking pairs of Cys shown to be neighbors will be determined electrophysiologically in inside-out patches from cells treated with crosslinkers. The tethering by covalent crosslinking of alpha SO-S6 to betal TM1 or TM2 should profoundly affect function if the alpha TMs normally move during gating, possibly altering alpha -beta interactions. The rate constants for function-altering crosslinking of pairs of Cys will be determined in the open state of the channel in outside-out patches and compared to the rate constants determined in the closed state. Differences in rate constants in the two states will indicate which TMs of alpha move relative to TMs in beta during gating. BK channels play a major role in the regulation of contractile tone in smooth muscle and neuronal function, and their pathophysiology is implicated in stroke, hypertension, and cardiovascular disease. Greater understanding of the molecular mechanisms of BK channel regulation will lead to improvedtherapeutics.
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