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?

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.

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.