1984 — 1986 |
Brink, Peter |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Junctional Membrane Conductance Regulation |
0.915 |
1985 — 1993 |
Brink, Peter R |
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. |
A Model of the Intercalated Disc of Myocardium @ State University New York Stony Brook
The intercalated disc of mammalian myocardium contains nexuses or gap junctions (A type). Do these junctions show voltage dependent behavior? This study proposes to use an experimental model system, the septate axon of the earthworm, to voltage clamp junctional membranes under varied conditions. This study proposes to distinguish voltage - dependent junctional membrane conductance changes from pH dependent changes. The homogeneity of junctional membrane function will also be tested by measuring junctional conductance first in the absence of large probes and then with them present. Will the probes decrease junctional conductance? If so, to what extent? The methods given in this proposal will answer these questions. The double voltage clamp is a useful tool in maintaining steady transjunctional potentials while uncoupling cells and will allow uncoupling to be studied without any changes in transjunctional voltage.
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1 |
1985 — 1988 |
Brink, Peter R |
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. |
Intercellular Communication @ State University New York Stony Brook
The purpose of this study is to monitor the structure of cell junctions (nexus or gap junction) under various conditions and relate that information to the conductance and permeability of the structure. The parameters to be manipulated are intracellular pH, temperature and solvents (D2O vs. H2O). Hopefully experiments like these will illuminate the suspected intercellular channel.
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1 |
1992 |
Brink, Peter R |
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. |
Model of the Intercalated Disc of Myocardium @ State University New York Stony Brook
The aim of the proposal is monitoring of gap junction channel activity. A preparation has been developed which allows exposure of cytoplasmic surfaces of membranes that contain gap junctions. The septal membranes of the extracellular clamp methods are used to allow monitoring of gap junction channels. The specific aims are: (1) Use the patch clamp to record single channels from an exposed cytoplasmic surface when the exposed membrane is known to contain gap junctions. (2) Determine if channel behavior is consistent with known properties of gap junctions. (3) Study the ultrastructure of the dissected preparation to determine if there are any morphological changes in gap junctions. (4) Determine the effects of H+, Ca++ and other agents on channel activity. (5) Study the effects of solvent exchange on the conductive properties of the channels. (6) Determine the selectivity sequence. (7) Use the double voltage clamp as a means of measuring macroscopic behavior.
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0.972 |
1992 — 1995 |
Brink, Peter R |
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. |
Plasma Cells in the Lacrimal Gland--Ig Secretion @ State University New York Stony Brook
DESCRIPTION (Investigator's Abstract): Tears, the secretory product of the lacrimal gland, serve not only as a lubricant of the corneal surface but also as a primary defense against airborne antigens that contact the avascular cornea. The major anti-bacterial/viral agents in tears are the immunoglobulins secreted by plasma cells of the lacrimal gland. Elucidation of the physiology of the lacrimal glans at the cellular level is of critical importance to the amelioration of conditions such as "dry eye." The major focus will be determination of the role membrane channels and receptor mediated messenger systems play in the modulation of immunoglobulin secretion. There is evidence to support the notion that plasma cells contain receptors responsive to neurotransmitters and that these same transmitters can alter antibody secretion by the lacrimal gland. The Harderian (lacrimal) gland of the chicken will be the system used because the gland has a very high density of plasma cells and the principle immunoglobulin secreted by the avian lacrimal gland is an IgG which is thought to diffuse passively and unmodified through the surrounding cortical epithelium (Mullock et al., 1981). This is distinctly different than the rat lacrimal gland where the secreted immunoglobulin (IgA) is dimerized and then secreted by the secretory epithelium (Peppard and Montgomery, 1987). The preliminary results indicate that there are receptor mediated mechanisms capable of modulating the secretory rate of IgG out of the lacrimal gland and that the receptors are housed within the membrane of the plasma cells of the gland. Electrophysiological data indicate that membrane potential (manipulated by maxi-K channel activity) is an intrinsic "down" regulator of immunoglobulin secretion.
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0.972 |
1995 — 1998 |
Brink, Peter R |
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. |
Cx43 and Cx37 Gap Junction Channel Activity @ State University New York Stony Brook
This proposal is focused on elucidating the gating mechanisms of connexins 43 and 37 under defined ionic and expression constraints. The aims are: 1) Determine the gating characteristics of a non-transfected population of connexin43 derived gap junction channels using dual whole cell batch clamp (DWCP). Human vascular smooth muscle-cell pairs (HVSM) derived from corpora cavernosa, which are known to contain connexin43, will be used as well as NRK cell pairs which contain rat connexin43. Gap junction channel gating will be monitored by measurement of the mean open time(s), mean closed time(s), open probabilities and voltage sensitivity. Further, we ascertain whether there is cooperative gating (non-independent gating between gap junction channels. 2a) Use DWCP in N2A cells transfected with rCx43 or humanCx37 and determine the behavior of heterotypic Cx43/Cx37 gap junction channels. Homotypic behavior for Cx37 is in the literature and we present data in the preliminary results for rCx43 transfected cells. We will continue to study the homotypic forms and determine their gating characteristics for comparison with non-transfected rCx43. Homotypic refers to two identical hemichannels linked to form a gap junction channel (Cx43/Cx43) and a heterotypic refers to two non-identical hemichannels linked together (Cx43/Cx37) where each hemichannel is composed of only one connexin type. 2b) Co-transfect Cx43 and Cx37 into N2A cells (a potential model for endothelial cell intercellular communication) and monitor the multichannel behavior. The low number of channels formed in the N2A cells in combination with our analysis will reveal any gating differences between homotypic and heterotypic gap junction channels as well as gap junctions made of heteromers (heteromers=hemichannels of two different forms,Cx43 and Cx37 found in the same hemichannel). Our assumption is that heteromeric and heterotypic forms are distinguishable from each other and homotypic forms in terms of parameters such as open probabilities, mean open times, mean closed times, cooperative gating, voltage dependence and unitary conductance. 3.) Use direct patch clamp on HVSM cells and transfected N2A cells to identify gap junction channels on the surface of freshly isolated cells. 4) Elucidate gate-to-gate interactions (contingent gating) for single gap junction channels using DWCP data where large deltaVj steps are applied and modeling both macroscopic currents and unitary channel activity. We will also use osmotic loading (Zimmerberg and Parsegian, 1986) to distinguish volume changes between different conductance states of gap junction channels.
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0.972 |
1997 — 2001 |
Brink, Peter R |
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. |
Permselectivity Properties of Connexins @ State University New York Stony Brook
DESCRIPTION: This grant application is aimed at establishing a link between the fine structure of the gap junction channel wall and channel permselectivity. The investigators propose to study the properties of rat Cx26, Cx32, Cx37, Cx40, Cx43 and Cx46 when expressed in transfected N2A cells. They will analyze the spread of anionic fluorescent probes via estimations of three basic parameters: cytoplasmic diffusion coefficient, junctional membrane permeability and leak permeability. The second specific aim is to transfect mouse N2A cells with mutant versions of the connexins studied under Specific aim 1, and to evaluate the same permeability parameters. Through these studies, the investigators propose to locate the site(s) that constitute the anionic filter. The mutagenesis analysis will focus on the four transmembrane domains, and the two extracellular loops. Under specific aim three, the applicants will use the dual whole cell patch clamp technique to verify the existence of gap junction channels and, if channels are found, to determine the unitary conductance and the cation/anion permeability properties of the mutant under study. Finally, under specific aim four, the investigators propose to characterize a relation between channel properties and dye transfer. The latter may allow for determination of permeability of large anions at the channel level.
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0.972 |
2002 — 2005 |
Brink, Peter R |
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. |
Gap Junction Channel Permeability: a Mutagenic Approach @ State University New York Stony Brook
DESCRIPTION (provided by applicant): We hypothesize that there are specific charge groups that preferentially influence the permeation of molecules in the size and charge range of second messengers while having little influence on monovalent cation selectivity. We hypothesize that there are multiple sites able to affect the permselectivity of gap junction channels. The specific aims are: Aim la) Transfect mouse N2A cells or rat insulinoma cells (RIN) with mutant versions of Cx43, Cx40, and Cx37 cDNA and determine expression via Western and Northern blot analysis. We will also use the GFP tag strategy to determine cellular distribution. We have chosen Cx43 and Cx40 because of their ubiquity in the SA an AV nodes of the heart and hence their potential to affect cardiac arrhythmias. Cx37 is the major connexin of endothelium. Aim ib) Monitor single channel conductance in homotypic mutants in KC 1, CsCI, NaCI and TEACI to assess changes in cation/anion selectivity using dual whole cell patch clamp. Aim 2) Monitor single channel conductance in which one hemichannel is composed of a mutant connexin (muCx43 or muCx4O) and the other is composed of a wild type connexin of Cx43 or Cx40 in Kcl, CsCI, NaCl and TEAC1 to assess changes in cation/anion selectivity. We will use cysteine scanning mutation to determine if a mutated site lines the poor wall. Aim 3) Simultaneously determine junctional conductance and junctional permeability to charged fluorescent probes for wild type and mutant connexins (homotypic and heterotypic forms). This will allow the determination of the permeation rates of charged probes in the 0.8-1.2 nm size range relative to K+/Cs+. Inert probes (Lucifer Yellow, DAPI, carboxyfluorescein) are probes of choice because they are not rapidly removed from the cytosol, as is the case for lP3 (Tau less than 60s).
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0.972 |
2003 |
Brink, Peter R |
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. |
Cardiovascular Role of Sympathetic K+ Channel Genes @ State University New York Stony Brook
Sympathetic neuronal activity regulates such cardiovascular variables as heart rate, arterial blood pressure and myocardial contractility. The excitability of sympathetic neurons is determined in large part by a conductance known as the M-current. These investigators have recently determined that the M-current is encoded by the KCNQ2 and KCNQ3 genes. Increased understanding of these genes is a prerequisite to further investigation of 1) how the M-current is regulated during sympathetic activity, 2) the developmental control of this current and 3) the targeting of the M-current, and the pathways involved in its regulation, for pharmacological interventions in cardiovascular pathologies. The overall goal of the present application is to understand at a molecular level the subunit interactions that generate the constituent channels of the M-current. The projects outlined in Specific Aim 1 use knock-out and gain-of-function experiments to confirm our hypothesis that the M-current of sympathetic neurons is encoded by KCNQ2 and KCNQ3. Specific Aim 2 would test the hypothesis that the formation of the M-current involves protein interaction between the KCNQ2 and KCNQ3 subunits. Specific Aim 3 would test the corollary that protein interaction between the subunits facilitates the movement of subunit proteins from the endoplasmic reticulum to the cell membrane. Specific Aim 4 would test the hypothesis that specific splice variants of the KCNQ2 and KCNQ3 genes encode the M-channel of sympathetic neurons.
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0.972 |
2003 — 2006 |
Brink, Peter R |
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. |
Water Transport in the Lacrimal Gland @ State University New York Stony Brook
DESCRIPTION (provided by applicant): Fluid secreted by the lacrimal gland is an essential component of tears and is estimated to contribute approximately 50% of the liquid volume bathing the cornea (Walcott, 1998). Recently in situ measurements of fluid production from the lacrimal glands of mice have demonstrated stimulated flow rates of 0.2-0.6 uL/minute (Walcott et al., 2002; Paranyuk et al., 2001; Moore et al, 2000). Our preliminary data indicates that the NKCC1 Na+, K+, 2CI- co-transporter and Bkca channels play important roles in fluid production of the lacrimal gland. We propose the use of knockout mice along with NZB mice (compromised fluid flow, Paranyuk et al., 2001) to study their respective roles in fluid production by the lacrimal gland. Further we propose the use of activators and inhibitors of PKC to delineate its role in the regulation of the NKCC1 co-transporter and Bkca channel. Both systems have been shown to be influenced/moduate/regulate by PKC (Standen and Quayle, 1998;Zhou et al., 2001; Clerice etal., 1995). Our proposal is focused on addressing the following hypotheses: Aim 1 Hypothesis: Basolateral blockade of the salt co-transporter (NKCC1) will significantly reduce stimulated fluid flow in controls but will be similar to NKCC1 knockout flow rates. We will also compare and contrast control and knockouts with NZB. We propose the use of NZB because our preliminary results indicates that the NZB acinar cells have significantly less amounts of NKCC1 than controls. Aim 2: Hypothesis: Apical membrane BKca channels contribute to normal fluid production of the lacrimal gland. We will test control (C57) and Bkca Beta 1 knockout mice. Aim 3: Hypothesis: PKC activity affects fluid production via regulation of K channels and/or NKCC1 transporters. We will test controls, NZB and the two knockouts (Beta1 and NKCC1).
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0.972 |
2010 — 2013 |
Brink, Peter R Cohen, Ira S (co-PI) [⬀] Mathias, Richard T (co-PI) [⬀] |
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. |
Sirna Therapeutics: Gap Junction Delivery in Vitro and in Vivo @ State University New York Stony Brook
DESCRIPTION (provided by applicant): The cellular delivery of siRNA via gap junctions represents a unique and potentially clinically important delivery system. Our previous studies have shown that gap junctions composed of connexin43 (Cx43) are permeable to siRNAs and permeating siRNAs can subsequently reduce the mRNA levels of a specific gene. The aim of the studies proposed here is to characterize the transfer and permeation of siRNA from hMSCs and other communication competent cells into a target tissue. Previously we determined that gap junctions composed of Cx43 transfer siRNA, whereas those composed of Cx32 or Cx26 will not. Hence channel permeability for siRNA depends on the connexin. In aim 1 we will determine the permeability of gap junctions made of Cx40, Cx37 and Cx43, to morpholinos and siRNAs. Cx43, Cx40 and Cx37 are chosen because they are ubiquitously expressed in vivo in many organs. In aim 2 we will determine the synthesis and degradation rates of siRNA targeting HCN2 and GFP. We will also investigate the efficacy and time course of functional silencing of the membrane protein, the pacemaker channel HCN2. We will test the hypothesis that cellular delivery of siRNA via gap junction channels can silence HCN2 channel function in target cells by characterizing the functional silencing of HCN2 via gap junction mediated delivery of siRNA from hMSCs or other communication competent cells to a target cell expressing HCN2. We follow HCN2 mRNA concentration using RT-PCR to allow an estimate of the relative content over time in the presence of siRNA. We will also determine the concentrations of tagged morpholinos/siRNAs to establish the effective concentration necessary to silence a gene (HCN2 or GFP) and also provide parameters for our 2D/3D model to determine penetration within a tissue. In aim 3 we will experimentally assess how far siRNA can penetrate multiple cell layers of a syncytium. In aim 4 we will derive a model for transfer of siRNA along a simple linear chain of cells or geometries in 2 or 3 dimensions. It will be used to predict the number and position of siRNA containing cells (hMSCs) required to silence function in a tissue or an organ/tumor. In aim 5 we will assess siRNA effectiveness in silencing GFP in vivo. We use nude mice and inject a bolus of 10 million cells expressing GFP into the dermis or intramuscularly followed at various times with an injection of hMSCs loaded with siRNA targeting GFP. We will track the GFP fluorescence image over time using whole animal imaging. PUBLIC HEALTH RELEVANCE: Small interfering RNA (siRNA) targets a single protein reducing its expression. As such it has great potential as a highly selective drug. However systems for its in vivo delivery are not optimal. The present application investigates the ability of the immuno-privileged adult mesenchymal stem cell (MSC) as well as other cell types to deliver small interfering RNA (siRNA) to a target cell or tissue. The basis of this cell based delivery is the gap junction channel. These channels connect the intracellular compartments of coupled cells and allow transfer of small molecules without entry into the extracellular space. We have already established that cells that make connexins (the building block of gap junctions) can transfer siRNAs. This application asks whether cells can serve as a delivery system for siRNA. By a combination of experiment and mathematical modeling we seek to determine the ability of cellular delivery of siRNA to penetrate tissues in vitro and in vivo.
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