Jonathan B. Cohen, Ph.D. - US grants

peptide chemical synthesis; biomedical facility; synthetic protein; high performance liquid chromatography; polymerase chain reaction;

This application for a Jointly Sponsored Predoctoral Training Program in Neurosciences will represent the major source of support for students in the Ph.D. Program in Neurosciences at Harvard University. The goals of this interdepartmental Ph.D. Program, which was established in 1981, are (1) to organize within a single training faculty the neuroscientists at Harvard Medical School, its affiliated hospitals, and Harvard College; in order (2) to train research scientists/teachers who have a broad background in neuroscience and who are interested in mental health and diseases of the nervous system to carry out original and rigorous research in important areas of neuroscience. In the first two years trainees complete a sequence of core courses ranging from cell and molecular neurobiology to systems neuroscience, as well as collateral courses selected from cell and molecular biology, immunology, statistics, and other subjects appropriate to individual interests. Students rotate through three different laboratories. Full time thesis research follows the course work, laboratory rotations, and qualifying exams. Students are also involved in other training activities including journal clubs, seminars and data presentation. There are currently 72 graduate students in the Program. The total faculty includes 94 members, of whom 54 who are currently most actively involved in graduate education are Training Mentors on the present grant. Considerable effort has gone into making this program a highly interactive group with extensive formal and informal contacts between students and faculty.

DESCRIPTION: (adapted from Applicant's Abstract) The long range goal of this research is to understand, in molecular terms, how synapses form during embryonic life and how they are maintained in adult animals. These events can be studied with great precision at the vertebrate neuromuscular junction, a synapse whose functional, morphological and biochemical phenotype is understood better than any other chemical synapse. This proposal is focused on ARIA, a polypeptide that was purified based on its acetylcholine receptor inducing activity in embryonic skeletal myotubes. ARIA probably acts at developing and mature junctions to promote the synthesis of ACh receptors and their accumulation in the postsynaptic muscle membrane. One immediate goal of the proposal is to study the processing of ARIA: how the synthesis is regulated in motor neurons, how it is transported to the motor axon terminals and how it is displayed in the synaptic cleft. Membrane binding sites will be studied in detail to determine which type correlates best with physiological responses. Another goal is to characterize ARIA binding proteins in the muscle membrane, and other binding proteins in the extracellular matrix that may modulate its action. Another important set of experiments is designed to define ARIA's spectrum of action at various stages of endplate development. This potent differentiation factor may regulate several genes that encode ion channels in the presynaptic and postsynaptic membrane, proteins in the postsynaptic cytoplasm, and proteins that are destined for the extracellular matrix. It may, in short, be a master switch that regulates synapse specific gene expression. ARIA is synthesized as a transmembrane precursor (proARIA), and several alternatively spliced isoforms have been uncovered. A final goal of this research is, therefore, to determine exactly which isoforms are expressed in motor neurons and the precise role for each one. The planned experiments depend on recombinant DNA, biochemical, electrophysiological and morphological techniques. Identification of ARIA as one of the first trophic factors operating at the neuromuscular junction opens new areas of study regarding the two-way, nutritive relationship between motor neurons and their synaptic partners in the periphery. ARIA was purified from the brain, and it is likely that it exerts powerful trophic effects in the central nervous system as well as in the periphery. Judging from its location in cholinergic neurons throughout the nervous system and in some non-cholinergic cells, our studies at the neuromuscular junction will have immediate import for studies of normal and abnormal development in CNS.

binding sites; nicotinic receptors; local anesthetics; drug interactions; neurotransmitter antagonist; receptor sensitivity; barbiturates; progestins; progesterone; lipid bilayer membrane; alcohols; halothane; progesterone analog; receptor expression; Xenopus oocyte; alternatives to animals in research; affinity chromatography; protein sequence; Torpedo;

DESCRIPTION (provided by applicant): We propose to continue a Jointly Sponsored Predoctoral Training Program in Neurosciences that is the major source of support for students in the Ph.D. Program in Neurosciences at Harvard University. The goals of this interdepartmental Ph.D. Program, which was established in 1981, are (1) to organize within a single training faculty the neuroscientists at Harvard Medical School, its affiliated hospitals, and Harvard College; in order to (2) to train research scientists/teachers who have a broad background in neuroscience and who are interested in mental health and diseases of the nervous system to carry out original and rigorous research in important areas of neuroscience. In the first 18 months trainees complete a sequence of core courses ranging from cell and molecular neurobiology to systems neuroscience, as well as collateral courses selected from cell and molecular biology, immunology, statistics, and other subjects appropriate to individual interests. Students rotate through three different laboratories. Full time thesis research follows the course work, laboratory rotations, and qualifying exams. Students are also involved in other training activities including journal clubs, seminars and data presentation. There are currently 76 graduate students in the Program. The total faculty includes 94 members, of whom 61 who are currently most actively involved in graduate education are Training Mentors on the present grant. Considerable effort has gone into making this program a highly interactive group with extensive formal and informal contacts between students and faculty.

LOCATING GENERAL ANESTHETIC BINDING SITES IN GABAA AND ACETYLCHOLINE RECEPTORS Our goal is to determine the number and location of the binding sites for general anesthetics in GABAARS, a major site of action of a structurally diverse group of general anesthetics used clinically, and to compare them with their binding sites in nicotinic acetylcholine receptors (AcChoRs), receptors that are both members of the "Cys-loop" superfamily of neurotransmitter-gated ion channels. Most anesthetics potentiate the action of GABA at the inhibitory GABAA receptors, while they inhibit the excitatory AcChoRs in brain and skeletal muscle. Within a single receptor multiple potential anesthetic binding sites are present in the pockets that exist within receptor subunits and at subunit interfaces, including the ion channel. It is our hypothesis that each anesthetic, depending on structural class, will interact preferentially with different receptor sites. An improved understanding of the diversity of general anesthetic binding sites within neurotransmitter-gated ion channels will allow for the design of safer anesthetic agents. We propose to use photoaffinity labeling and protein chemistry techniques to identify the binding sites for photoreactive aliphatic alcohols and analogs of etomidate, propofol, and barbiturates in GABAARS isolated from detergent extracts of bovine brain and cultured cells, in neuronal a4p2 AcChoRs isolated from cultured cells, and in muscle-type AcChoRs in nicotinic postsynaptic membranes isolated from Torpedo electric organ. We will determine whether a single anesthetic binding site in a GABAAR (or AcChoR) can be occupied by drugs that, depending on structure, act as potentiators or inhibitors. These photoaffinity labeling studies make use of the Synthetic Chemistry, Protein Chemistry and Protein Production Cores. Our structural studies are complementary to the time- resolved photolabeling studies in (Miller/Raines Project) and to the mutational analyses (Forman Project) designed to determine whether the sites we identify are sites of functional potentiation or inhibition. RELEVANCE (See instructions): GABAARS in the brain are a major site of action of many clinically used anesthetics of diverse chemical structure, but the number and location of anesthetic binding sites within GABAARS or in the structurally related nicotinic acetylcholine receptors remains unknown. Identification of these sites will contribute to the development of anesthetics with greater selectivity and fewer side-effects.

CORE C: Protein Chemistry Core The Protein Chemistry Core will continue to provide for the research groups in the Program Project the technical expertise and equipment required for the isolation and sequence analysis of receptor fragments containing the sites of photoincorporation by general anesthetics. In addition, the Core provides computational resources for the molecular modeling that is necessary to interpret the protein chemistry results in terms of models of receptor structure and to provide guidance in the design of mutational analyses necessary to define the energetic contributions to anesthetic binding. The Core is under the supervision of Dr. Cohen and is located in his laboratory. Major equipment cunrently available in the Core include (i) an Applied Biosystems 492 Precise automated Protein Sequenator and in-line amino acid analyzer;and (ii) 2 Agilent 1100 HPLCs equipped with UV and fluorescence detectors;and (iii) an Agilent 1100 capillary LC system. The biochemical characterization of anesthetic binding sites in ligand-gated ion channels requires the use of highly specialized techniques not normally available in protein chemistry core facilities which usually do not accept radioactive samples either for Edman degradation or mass spectrometry. The identification of drug binding sites within the hydrophobic domains of integral membrane proteins poses unique problems for peptide isolation and characterization by either Edman degradation or mass spectrometry. It is the function of the Core to provide the appropriate equipment and highly skilled protein chemists who can interact directly with the research projects to develop and carry out the appropriate research strategies and to educate the investigators about the necessary protein chemistry techniques to be carried out either in the Core or in the investigator's lab. RELEVANCE (See instructions): GABAARS in the brain are a major site of action of many clinically used anesthetics of diverse chemical structure, but the number and location of anesthetic binding sites within GABAARS or in the structurally related nicotinic acetylcholine and serotonin 5-HT3 receptors remains unknown. This Core provides the resources necessary to identify these binding sites.

LOCATING GENERAL ANESTHETIC BINDING SITES IN GABAA AND ACETYLCHOLINE RECEPTORS Our goal is to determine the number and location of the binding sites for general anesthetics in GABAARS, a major site of action of a structurally diverse group of general anesthetics used clinically, and to compare them with their binding sites in nicotinic acetylcholine receptors (AcChoRs), receptors that are both members of the Cys-loop superfamily of neurotransmitter-gated ion channels. Most anesthetics potentiate the action of GABA at the inhibitory GABAA receptors, while they inhibit the excitatory AcChoRs in brain and skeletal muscle. Within a single receptor multiple potential anesthetic binding sites are present in the pockets that exist within receptor subunits and at subunit interfaces, including the ion channel. It is our hypothesis that each anesthetic, depending on structural class, will interact preferentially with different receptor sites. An improved understanding of the diversity of general anesthetic binding sites within neurotransmitter-gated ion channels will allow for the design of safer anesthetic agents. We propose to use photoaffinity labeling and protein chemistry techniques to identify the binding sites for photoreactive aliphatic alcohols and analogs of etomidate, propofol, and barbiturates in GABAARS isolated from detergent extracts of bovine brain and cultured cells, in neuronal a4p2 AcChoRs isolated from cultured cells, and in muscle-type AcChoRs in nicotinic postsynaptic membranes isolated from Torpedo electric organ. We will determine whether a single anesthetic binding site in a GABAAR (or AcChoR) can be occupied by drugs that, depending on structure, act as potentiators or inhibitors. These photoaffinity labeling studies make use of the Synthetic Chemistry, Protein Chemistry and Protein Production Cores. Our structural studies are complementary to the time- resolved photolabeling studies in (Miller/Raines Project) and to the mutational analyses (Forman Project) designed to determine whether the sites we identify are sites of functional potentiation or inhibition. RELEVANCE (See instructions): GABAARS in the brain are a major site of action of many clinically used anesthetics of diverse chemical structure, but the number and location of anesthetic binding sites within GABAARS or in the structurally related nicotinic acetylcholine receptors remains unknown. Identification of these sites will contribute to the development of anesthetics with greater selectivity and fewer side-effects.

Project Summary - Project 1 (Locating General Anesthetic Binding Sites in GABA-A and Glycine Receptors) Our broad goal is to determine the locations in human GABAA receptors (GABAAR) of the binding sites for general anesthetics of diverse chemical structures and the affinities of anesthetics for the different classes of sites. In the previous grant period, we used photoreactive etomidate and barbiturate analogs to establish for the first time that there are at least two classes of intersubunit general anesthetic binding sites in the transmembrane domain of a human ??? GABAAR. One class is located at the interfaces between subunits containing the transmitter binding sites in the extracellular domain and the second class at interfaces not containing the transmitter binding sites. Etomidate binds at anesthetic concentrations with >100-fold selectivity to the first class of sites, and certain barbiturates bind with high selectivity to the second class. We also determined that propofol binds non-selectively to both sites, but only at concentrations greater than necessary to produce anesthesia or potentiate GABA responses, and anesthetic steroids do not bind to either class of sites. Our first aim for this renewal is to use novel photoreactive analogs of anesthetic steroids, barbiturates, and propofol to identify in expressed, human ?1?3?2 GABAAR additional anesthetic binding sites important for GABAAR potentiation or inhibition. It is our hypothesis that anesthetic steroids bind to intrasubunit sites at the interfaces between transmembrane helices that are also in contact with lipid. A photoreactive analog of a convulsant barbiturate will be used to identify novel binding sites important for GABAAR inhibition. Photoreactive propofol analogs will be used to determine whether propofol binds in a GABAAR to intrasubunit sites equivalent to those identified previously in nicotinic acetylcholine receptors and a prokaryote GABAAR homolog. We will determine in Aims 2 and 3 whether anesthetics bind to equivalent sites in a GABAAR subtype that is expressed extrasynaptically (?4?3d) and in glycine receptors, the receptor most similar in structure to GABAARs and the major inhibitory receptor in the spinal cord. The proposed experiments make use of the Synthetic Chemistry, Protein Chemistry, and Protein Production Cores. Our studies, in conjunction with the development and characterization of novel, potent anesthetics and time-resolved photolabeling studies of Project 2 (Miller) and the functional studies of Project 3 (Forman), should provide an unprecedented view of the number and diversity of general anesthetic binding sites in GABAARs and provide a starting point for the development of anesthetics selective for GABAAR subtypes.

Project Summary - Core C (Protein Chemistry Core) The Protein Chemistry Core will continue to provide the research groups in the Program Project with the technical expertise and equipment required for the isolation and sequence analysis of receptor fragments containing the sites of photoincorporation by general anesthetics. In addition, the Core provides computational resources for the homology modeling and docking studies that are necessary to interpret the protein chemistry results in terms of models of receptor structure (Projects 1 and 2) and to provide guidance in the design and interpretation of mutational analyses (Project 3) that further assess the structure and function of the identified binding sites. The Core is under the supervision of Dr. Cohen and is located in his laboratory. Major equipment currently available in the Core include (i) an Applied Biosystems 492 Procise automated Protein Sequenator and in-line amino acid analyzer; and (ii) 2 Agilent 1100 HPLCs equipped with UV and fluorescence detectors; and (iii) an Agilent 1100 capillary LC system. The biochemical characterization of anesthetic binding sites in ligand-gated ion channels requires the use of highly specialized techniques not normally available in protein chemistry core facilities which usually do not accept radioactive samples either for Edman degradation or mass spectrometry. The identification of drug binding sites within the hydrophobic domains of integral membrane proteins poses unique problems for peptide isolation and characterization by either Edman degradation or mass spectrometry. It is the function of the Core to provide the appropriate equipment and highly skilled protein chemists who can interact directly with the research projects to develop and carry out the appropriate research strategies and to educate the investigators about the necessary protein chemistry techniques to be carried out either in the Core or in the investigator's lab. In the previous funding period, novel experimental strategies were developed to characterize anesthetic photolabeling of amino acids in each of the transmembrane helices of the a1?3g2 GABAAR, which led to the identification of a second class of GABAAR anesthetic binding sites, and in serotonin 5-HT3 receptors. This Core will now provide similar expertise for the characterization of novel anesthetic binding sites in a1?3g2 and a4?3d GABAARs and GlyRs.