1989 — 1997 |
Peralta, Ernest G |
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. |
Signal Transduction Mechanisms of Muscarinic Receptors
Muscarinic acetylcholine receptors (mAChRs) are a family of at least four structurally distinct subtypes which regulate a variety of physiological and biochemical responses throughout the central and autonomic nervous systems. The proposed research will employ techniques of molecular genetics to study the interactions between recombinant mAChR subtypes, the adenylyl cyclase and phosphoinositide biochemical effector pathways, and ion channels in tissue culture expression systems. These studies will provide important information regarding the mechanisms by which individual mAChR subtypes interact with G proteins to regulate central nervous activities and control the rate and force of heart muscle contraction. The structural domains involved in determining the specificity of G protein activation by mAChR subtypes are unknown. To determine the regions of these receptors involved in G protein recognition and activation, a series of hybrid receptors composed of domains derived from functionally distinct Ml and M2 mAChR subtypes will be constructed and expressed within stably transfected mammalian cell lines. The Ml subtype potently stimulates PI hydrolysis and fails to inhibit adenylyl cyclase, while the M2 subtype efficiently inhibits adenylyl cyclase and weakly increases PI hydrolysis. Each hybrid receptor will be tested for its ability to bind muscarinic ligands, inhibit adenylyl cyclase, stimulate PI hydrolysis, and release intracellular calcium. The effects of pertussis toxin on these responses will also be investigated. These studies will identify important domains through gain, rather than loss, of function. The mechanisms of ion channel regulation by individual mAChR subtypes are poorly understood. To determine the ability of individual subtypes to regulate ion channel activity, molecular clones encoding acetylcholine-sensitive potassium channels will be isolated and expressed in the presence of defined mAChR subtypes and G proteins. In particular, molecular clones encoding the atrial inward rectifying potassium channel will be isolated to study the novel regulation of this channel by mAChRs. The effect of muscarinic stimulation on channel activity will be analyzed by patch clamp of transfected mammalian cells and voltage clamp of Xenopus oocytes injected with mRNAs encoding potassium channel and mAChR proteins. Ultimately, mutagenesis studies will be conducted to determine the important structures involved in the regulation of potassium channels by mAChRs and G proteins.
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1 |
1991 — 1994 |
Peralta, Ernest G |
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 Regulation of Chloride Ion Channels
At the cellular level, the principal defect in cystic fibrosis (CF) appears to be the abnormal regulation of Cl- transport across the apical membrane of airway epithelia. Apical CI- channels on the epithelia of CF patients are insensitive to stimulation by the Ca 2, or cAMP- dependent pathways which activate Cl- conductances in normal cells. Although the most common genetic defect in CF occurs within the cystic fibrosis transmembrane regulator (CFTR), the relationship between this protein and the regulation of apical Cl- conductances is unknown. The proposed research will employ techniques of molecular genetics and electrophysiology to isolate molecular clones encoding Cl- channels and study the regulatory interactions between Cl- channels and the cAMP and Ca2+ second messenger pathways. These studies will provide important information regarding the structure of Cl- channels and the mechanisms by which these channels are regulated in normal and CF afflicted cells. The primary structures of CI- channels remain unknown. To obtain molecular clones encoding CI- channels, size-fractionated mRNA, isolated from a cell line expressing these Cl- channels, has been identified which directs the expression of Cl- conductances following injection and two electrode voltage clamp of Xenopus oocytes. A cDNA expression library derived from this mRNA will be transcribed in vitro and similarly analyzed in oocytes: this will allow the identification of clones encoding Cl- channels.These studies will provide the first information regarding the primary structure and potential regulatory domains of Cl- channels. The molecular mechanisms by which Cl- channels are regulated are poorly understood. To determine the mechanisms of channel regulation, molecular clones encoding Cl- channels will be expressed alone or in the presence of cloned beta-adrenergic or muscarinic acetylcholine receptors The effect of beta-adrenergic stimulation (coupled to cAMP increase), muscarinic stimulation (coupled to Ca2+ increase) and membrane potential on Cl- channels will be analyzed by patch clamp of transfected mammalian cells or voltage clamp of Xenopus oocytes. In tandem, CF-affected skin fibroblast cells will be studied under patch clamp to assess the physiologic relevance of expression studies. Site-directed mutagenesis of the cloned Cl- channel will ultimately allow identification of the critical structures involved in channel regulation.
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1 |
1995 — 1997 |
Ptashne, Mark Strominger, Jack (co-PI) [⬀] Wang, James (co-PI) [⬀] Maniatis, Thomas Peralta, Ernest |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of a Surface Plasmon Resonance (Spr) Spectrometer
As a consortium of five faculty members and their research groups in the Department of Molecular and Cellular Biology, Harvard University, we propose the acquisition of a Surface Plasmon Resonance (SPR) Spectrometer. This instrument can detect the binding of biomolecules in solution to a surface-immobilized ligand. The SPR instrument is extraordinarily sensitive because it uses a laser at a precise angle to excite the surface plasmons at the glass-gold interface of the SPR chip (upon which the measurements are made). The change of angle of the reflected minimum of intensity as a function of time is correlated with binding of the biomolecule(s) in solution with the immobilized ligand. Using this machine, it is possible to measure association and dissociation rates of binding thereby providing both kinetic and thermodynamic data from the same measurements. With the recent addition of variable temperature capability entropic and enthalpic contributions to binding energy can be separated. We plan to follow several lines of research with the new instrument, pertaining to: (a) the mechanism of transcription; (b) the mechanism of the immune response; (c) signal transduction pathways; and (d) drug/target inhibitory effects. In parallel with the experiments mentioned above, we will develop new SPR technologies; in particular we plan to design new SPR chips as follows: We propose a new paradigm for surface immobilization of biomolecules: the use of mixed, self-assembled monolayers (SAMs) of thiols, a small percentage of which have been derivatized to target particular classes of biomolecules. We have already demonstrated the efficacy of this strategy for a chip which selectively immobilizes histidine-tagged proteins through binding to Ni(II). Use of the technique resulted in an enhanced sensitivity of at least an order of magnitude. In addition to further development of this chip, we plan to contruct a similar chip for binding biomolecules to immobilized DNA. Lastly, we propose the development of a chip to display an array of small drugs to potential putative target molecules.
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0.915 |