Robert L. Rosenberg, PhD - US grants

The activity of voltage-sensitive calcium channels is vital to the normal function nerve cells; Ca entry through these channels underlies bursting patterns, neurotransmitter release, neurite growth, and changes in protein phosphorylation. The functional properties several types of neuronal Ca channels have been described, but our understanding of their structures and of the molecular interactions responsible for channel activation, inactivation and modulation remains primitive' This proposal describes studies of neuronal Ca channnels isolated from their complex cellular environment. First, Ca channels from synaptosomes will reconstituted into planar bilayers and other simple membrane systems where their functional properties can be examined. Second, neuronal Ca channels will be solubilized and partially purified in order to study their protein structures. Reconstitution experiments will help answer some fundamental questions. Do the several types of neuronal Ca channels share key elements of ion selectivity and permeation cardiac Ca channels? Are the ion permeation pathways functionally symmetric like cardiac channels? What is the molecular mechanism of Ca-dependent inactivation of L-type channels? Can direct evidence for the role of cAMP-dependent phosphorylation in maintaining active Ca channels be obtained in the cell-free system? Can Ca channels in artificial membranes be modulated by phosphorylation or by direct interactions with purified G-proteins? These questions can be answered with reconstituted systems because of the easy experimental access to the internal side of the channel. The solubilization and isolation of Ca channels (as identified by the binding of a specific peptide neurotoxin w-conotoxin VIA) will provide information about the physical structure neuronal Ca channels. What is the peptide composition? How are neuronal w-conotoxin sensitive Ca channels different from skeletal muscle dihydropyridine-sensitive Ca channels? Are there immunological similarities? Are there important differences in carbohydrate composition? Is there evidence for direct interactions of modulatory proteins with the channels molecules. Are interactions with cell matrix proteins important for the localization and clustering of channels in the presynaptic membrane? Are w-conotoxin binding proteins functional, channels when they are reincorporated into membranes?

The objective of this proposal is to learn about the structures of the essential penicillin-binding proteins (PBPs) of Escherichia coli and to understand how the structures of these proteins relate to and affect their function in the synthesis of the cell wall peptidoglycan. Many questions still exist as to how these PBPs control such diverse processes as elongation, determination of cell shape,and cell division. Since PBPs are killing targets for B- lactam antibiotics, the need to learn more about their functions provides a compelling reason for the continuing study of these proteins. An understanding of the molecular basis of B-lactam recognition and peptidoglycan biosynthesis should contribute greatly to the rational design of new antibiotics. The specific aims of this proposal are: 1) to continue to supply protein for the solving of the crystal structure of a soluble form of a mutant PBP 5 (in collaboration with Dr. Brian Sutton, Kings College, London), 2) to construct expression systems for the overproduction and eventual crystallization of soluble PBPs 1A and 1B, 3) to construct chimeric proteins of PBPs 1A, 1B, 2 and 3 by fusing the transpeptidase (B-lactam-binding) domain of one PBP with the transglycosylase domain of another PBP, and use these chimeras to study the functions and interactions of the different domains by assaying their affects on cell morphology and viability, 4) to produce B-lactam-binding domains from any of the high molecular weight PBPs and use them for crystallographic studies as well as investigating the domain structures of these PBPs, and 5) to exchange the cytoplasmic and transmembrane regions of PBPs 1B, 2, and 3 with each other and assaying whether these regions have any function in the regulation of activity of the PBP. These experiments combine the techniques of molecular biology with those of biochemistry and crystallography. With these approaches, it is hoped that much useful information will be gained on the structural aspects of PBPs and how these structures relate to their observed function.

L-type Ca channels serve many vital functions in the heart. The influx of Ca2+ ions through L-type Ca channels during the plateau of the cardiac action potential provides the "trigger" for the release of Ca2+ from the sarcoplasmic reticulum. This establishes the link between electrical events at the cell surface and the initiation of contraction. The activation of L-type Ca channels in nodal cells contributes substantially to the pacemaker rates and to the speed of action potential propagation in those cells. Modulation of Ca channel activity is a crucial element in positive and negative inotropy and chronotropy, and may play a crucial role in the generation of very slow conduction and the development of the re-entry circuits in ischemic ventricular muscle that can lead to ventricular fibrillation. The long-term goal of this project is to characterize how changes in the intracellular environment affect L-type Ca channel activity. The objective it to understand the molecular forces that control Ca channel activity under normal conditions as well as those that are important during cardiac ischemia. Changes in the membrane potential and the intracellular concentrations of Ca2+ , Mg2+, H+, and ATP are known to result from ischemic episodes, and all impact on the availability, open probability, and gating kinetics of L-type Ca channels. In addition, the activity of intracellular proteins such as protein kinases, phosphoprotein phosphatases, lipases, and regulatory G-proteins also have strong effects on channel activity. The experimental approach is to characterize the effects of intracellular ions and regulatory proteins on L-type Ca channels incorporated from cardiac sarcolemma into planar lipid bilayers and in excised patches from giant reconstituted liposomes, where there is good experimental access to the intracellular side of the channel. We will test whether specific ionic or enzymatic modulators of Ca channels act directly on the channels or via intermediate effector systems. Endogenous regulatory components that are present in the sarcolemmal membranes will be activated or inhibited by specific drugs or ligands, and the effect of that modification on L-type Ca channel behavior will be studied. Additionally, exogenous enzymes, other proteins, ions, or small molecules will be added to the intracellular side, and the effect on channel properties recorded. Modification of Ca channel properties will be observed at the single-channel level as changes in conductance, open probability, activation or inactivation kinetics, open- and closed-time distributions, or "lifespan" of the channels in the cell-free membranes. In order to dissociate any multi-protein complexes of channels and endogenous regulators, we will also study the properties of Ca channels in membrane fragments stripped of extrinsic membrane proteins by treatment with high pH or with other chaotropic agents. In addition, Ca channels will be solubilized from the sarcolemma and reconstituted into artificial membranes, providing even better control of the channel's regulatory environment.

gene expression; transfection; biomedical facility; animal colony; genetically modified animals; laboratory mouse;

L-type Ca channels serve many vital functions in the heart. They sustain the plateau of the action potential and help set action potential duration and refractoriness. The influx of Ca2+ through L-type Ca channels triggers Ca2+ release from the sarcoplasmic reticulum, establishing the link between electrical activity at the cell surface and the contraction of cardiac myoctes. L-type Ca channels in nodal cells contribute to pacemaker activity and to the speed of conduction in those cells. Their activity may also play an important role in the generation of very slow conduction in ischemic myocardium, and especially at the border between normal and ischemic regions. In order to understand the basis of electrical re-entry that can occur at the ischemic border zone to trigger dangerous ventricular arrhythmias, it is crucial to understand factors that modulate Ca channels. The long-term goal of this project is to characterize the molecular mechanisms responsible for the modulation of cardiac L-type Ca channels, with special attention to the conditions that exist at the ischemic border zone. Because ischemic myocardium has a depolarized resting potential, high intracellular Ca2+, and low border zone. Because ischemic myocardium has a depolarized resting potential, high intracellular Ca2+, and low intracellular and extracellular pH, and because cardiac ischemia produces large beta-adrenergic stimulation, Specific Aim 1 focuses on the regulation of L-type Ca channels by protein kinase A under depolarized, Elevated Ca2+, and acidic pH conditions. We hypothesize that PKA stimulation sustains the activity of Ca channels the might otherwise become inactivated, leading to a abnormal slow conduction. Because beta- adrenergic release of norepinephrine is accompanied by a release of ATP, Specific Aim 2 focuses on some new observations of the effects of extracellular ATP on L-type Ca channels. Lipid metabolism is affected by ischemia, so Specific Aim 3 focuses on the regulation of L-type Ca channels by lysolipids and metabolites of arachidonic acid. Finally, Specific Aim 4 focuses on the regulation of Ca channels by nitric oxide and S-nitrosothiols. The experimental approach is to characterize the regulation of single L-type Ca channels incorporated from cardiac sarcolemma into planar lipid bilayers. We will record single channel activity of the reconstituted Ca channels, and evaluate channel conductance and unitary gating events. We control the extracellular and intracellular ionic conditions on both sides of the channels, the activity of endogenous regulatory enzymes like kinases and phosphatases, and the membrane environment. Thus, we will create conditions that mimic the ischemic border zone, and make a detailed characterization of the modulation of Ca channels there.

DESCRIPTION: The habit of smoking tobacco products creates major health problems in the U.S. Because of the significant behavioral and psychological effects of nicotine, this research focuses on nicotinic acetylcholine receptors (nAChRs) from the central nervous system. The goals of this research are (1) to characterize the permeation of calcium ions through neuronal nAChRs, (2) determine how intracellular Ca regulates the activity of neuronal nAChRs, and (3) evaluate how mutations of the pore-lining amino acids change ion permeation, receptor desensitization, and pharmacological properties. The research will focus on the alpha7 neuronal nAChR because they are localized at presynaptic terminals and are sensitive to a-bungarotoxin, suggesting their importance in regulating neurotransmitter release. Several complimentary experimental approaches will be used. AChRs will be expressed in Xenopus oocytes to evaluate channel viability, macroscopic receptor desensitization rates, and pharmacological properties. Alpha7 nAChRs will be reconstituted from oocyte surface membranes into planar lipid bilayer where we can control the ionic and regulatory environment of the channels. Single-channel electrophysiological approaches will be used to study (a) the affinity of permeant ion interactions with the pore, (b) the permeation of Ca through the pore, and (c) the regulation of the channel activity by intracellular Ca. In addition, nAChRs will be synthesized in vitro using a rabbit reticulocyte lysate system supplemented with endoplasmic reticulum microsomes. PAGE, density gradients, and ligand binding techniques will be used to study the biochemical properties of the synthesized AChRs. The in vitro synthesized nAChRs will be reconstituted into planar lipid bilayers to characterize their functional properties in the absence of other membrane proteins that may act as allosteric regulators. The detailed analysis of the biochemical and functional properties of several forms of alpha7 nAChRs will provide valuable information about these nicotinic receptors, the molecular basis of nicotine addiction, and perhaps the basis for the death of cholinergic neurons in Alzheimer's disease.

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms of agonist binding and agonist-dependent conformational changes during activation of neuronal nicotinic acetylcholine receptors (nAChRs) is essential for our understanding nicotine addiction, certain forms of epilepsy, Alzheimer's disease, and other neurological disorders. The research in this proposal focuses on alpha7 neuronal nAChRs because they are important in modulating neurotransmitter release. Specific residues that are important for ligand binding have been identified and characterized, but the conformational changes that couple agonist binding to channel gating are unclear. We use a homology model of the alpha7 receptors based on the crystal structure of the ACh Binding Protein to identify amino acids that may play important roles in agonist- or antagonist-dependent conformational changes. The specific goals of this research are to: (1) test the hypothesis that agonist binding causes a contraction or partial collapse of the ligand-binding pocket; (2) test the hypothesis that the beta9-beta10 hairpin structure, connecting the ligand-binding pocket to the transmembrane pore domain, undergoes conformational changes following the binding of agonists; and (3) determine whether lateral movement of the beta9-beta10 hairpin and/or rotational movements of subunits participate in agonist-driven activation. Several complementary experimental approaches will be employed. We will express alpha7 receptors in Xenopus oocytes and use electrophysiological approaches to probe functional properties. We will use the substituted cysteine accessibility method to identify residues that change accessibility during ligand binding. In addition, we will introduce electrostatic constraints and form disulfides or cysteine cross-links in (and near) the beta9-beta10 hairpin to limit movement and alter receptor activation.

DESCRIPTION (provided by applicant): Support is requested to renew and continue a broad, comprehensive and fundamental interdisciplinary predoctoral training program in the neurosciences at the University of North Carolina at Chapel Hill. The training will be administered by the interdepartmental Curriculum in Neurobiology which is in its third decade of existence as a Ph.D. degree granting entity. Training will involve 48 faculty members of the Curriculum representing research laboratories in 14 departments or programs. These research facilities are well equipped and funded for a wide variety of anatomical, molecular, genetic, biochemical, physiological, behavioral, and biophysical investigations. During the past year the Curriculum has become closely integrated with the new UNC Neuroscience Center providing expanded opportunities for training through the development of new research laboratories, the recruitment of new faculty, and occupancy of a new research building in the Fall of 2001. The formal training program is already in place and constitutes a series of required and elective learning activities leading to the Ph.D. in Neurobiology. Learning activities include formal coursework in a recently reorganized curriculum, laboratory apprenticeship research with individual faculty mentors, focused dissertation research, research seminars, techniques seminars, clinical correlation experiences, journal clubs, and discussion groups on topics of career development and research integrity. In addition an annual Carolina Neuroscience Symposium will expose trainees to current thought on specialized topics presented by distinguished scientists from outside UNC. An important central goal is to train individuals to utilize methods from a variety of disciplines (e.g. ultrastructure, molecular biology, molecular genetics, electrophysiology) to probe important problems in neurobiology. The proposed training program will take advantage of several areas of particular strength in neurobiology research at UNC including: (1) molecular and genetic control of neural development (2) molecular correlations of specific sensory neuronal function, (3) glial cell biology, (4) structure, function, and regulation of neurotransmitters, their receptors and transporters, (5) mechanisms of signal transduction and ion channel function, (6) neuroendocrine and neuroimmune interactions, (7) functional imaging of nervous system activity in vitro, in vivo, and in situ, and (8) distribution and regulation of neuropeptides active in various regions of the central nervous system. Support is requested for 10 predoctoral trainees. Qualified minority candidates will be aggressively recruited.