William A. Catterall - US grants

Previous work on this project has defined the purification and subunit composition of brain sodium channels, reconstituted their functional properties in purified form, and described their biosynthesis and assembly in neurons. cDNAs encoding Type IIA sodium channels have been cloned and expressed in mammalian cells. Regulation of sodium channel function through phosphorylation by cAMP-dependent protein kinase and protein kinase C has been analyzed in whole cell voltage clamp and single channel recording experiments. Structure-function studies with site-directed antibodies and site-directed mutagenesis have identified regions of the channel that are critical for voltage-dependent activation, fast channel inactivation, binding of alpha-scorpion toxins, and regulation of the channel by protein phosphorylation. The proposed research aims to build on this previous work to define several related aspects of sodium channel structure and function concerning mechanisms of channel inactivation regulation by protein phosphorylation, and modulation by drugs and neurotoxins. The functional role of individual amino acid residues in a inactivation gating loop will be probed by site- directed mutagenesis, expression in mammalian cells, and patch clamp recording; the mechanism of coupling of activation to inactivation will be analyzed through mutagenesis of amino acid residues in likely coupling segments. The sites of phosphorylation of sodium channel alpha subunits by protein kinase C and cAMP-dependent protein kinase that are responsible for down regulation of channel activity and modulation of channel inactivation will be identified by antibody mapping and protein chemistry, and the functional effects of their phosphorylation will be studies by mutagenesis expression, patch clamp recording, and biochemical analysis. Receptor sites for local anesthetics and sodium channel-directed anticonvulsant drugs on the intracellular surface of the sodium channel will be probed by mutagenesis and expression methods, and the molecular mechanisms of interaction of inactivation gating with the frequency- and voltage- dependent binding of these drugs will be analyzed. Receptor sites for alpha- and beta-scorpion toxins on the extracellular surface of the sodium channel will be identified by antibody mapping and protein chemistry, and their role in the normal coupling of activation to inactivation and the pharmacological effects of the toxins on these processes will be probed by mutagenesis and expression methods. The results of these studies will give new insight into the molecular mechanism of inactivation gating of sodium channels, its coupling to voltage-dependent activation, and its modulation by protein phosphorylation, local anesthetic and anticonvulsant drugs, and scorpion neurotoxins.

Voltage-sensitive calcium channels mediate calcium influx and calcium-dependent depolarization in response to changes of membrane potential in many excitable tissues. In the heart, calcium influx mediated by these channels is responsible for the plateau phase of the cardiac action potential and initiates excitation-contraction coupling. The positive inotropic and chronotropic of norepinephrine and other Beta-adrenergic agents mediated by cAMP-dependent protein phosphorylation is due in part to an increase in the number of calcium channels activated during the cardiac action potential. In this research, radiolabeled organic calcium antagonists such as [H3]-nitrendipine will be used as probes to monitor purification of the protein components of the calcium antagonist receptor of the voltage-sensitive calcium channel. The calcium antagonist receptor will be solubilized with digitonin and purified by conventional and affinity chromatographic procedures. Preliminary results show that the purified calcium antagonist receptor consists of a complex of three subunits. The purified receptor will be characterized with respect to subunit size, composition, stoichiometry, and general biochemical properties. The purified calcium antagonist receptor will be incorporated into phospholipid vesicles and its functional activity in ion transport will be assessed by isotopic flux measurements to determine whether the purified receptor contains all the components required to mediate voltage-sensitive calcium conductance. The subunits will be separated under native conditions and the functional role of the individual subunits assessed by reconstitution. Sites of phosphorylation of the subunits of the calcium channel will be identified both in vitro and in vivo and the functional effects of pholphorylation will be analyzed. The structure of the calcium channel will be investigated further by preparation and molecular cloning of cDNA encoding the channel subunits. These cDNA clones will then be sequenced and the amino acid sequence and secondary structure of the calcium channel subunits will be derived from the nucleotide sequence to give a detailed structural model of the calcium channel. These results will provide a basis for analysis of the molecular basis of calcium channel function and physiological regulation.

The Ninth Gordon Conference on Molecular Pharmacology will be organized around the theme of "Mechanisms of Membrane Signal Transduction", an area of rapid advance in research in molecular pharmacology. The conference is designed to promote interdisciplinary exchange of current ideas and information among participants with interests in a broad range of experimental problems related to this theme. Speakers using a wide range of experimental approaches in physiology, pharmacology, biochemistry and molecular genetics have been invited to participate. The speaker list includes both established and junior investigators from universities, research institutes and industrial laboratories. Participants will be selected to maximize audience contributions to discussion and to poster sessions. Broad representation of disciplines and institutions will be sought. Emphasis will be placed upon selection of women and young scientists as participants. Basic mechanisms of transmembrane signaling are repeated throughout biology. These processes underlie information processing and transfer in the nervous system and impulse initiation, condition and regulation in the heart. Insulin and other growth factors initiate their physiological effects by actions at the cell membrane which are transmitted by intracellular signals. Oncogenes are derived from growth factors, their receptors, membrane signal transduction proteins and their intracellular effectors, the protein kinases. Therefore, the topics to be considered cut across the areas of interest of NIGMS, NINCDS, NHLBI, NIADDK, and NCI. Fundamental knowledge of mechanisms of transmembrane signaling will provide a basis for new understanding of pathophysiology and therapeutic intervention in diverse disease entities including epilepsy and other neurological diseases, cardiovascular disease and cardiac arrhythmia, diabetes, and cancer.

Anticonvulsant drugs can be separated into groups based on their clinical efficacy against specific classes of seizures. Data in the literature and preliminary results presented herein suggest that diphenylhydantoin and carbamazepine which are primarily effective against partial and grand mal seizures act selectively on voltage-sensitive sodium channels; phenobarbital, benzodiazepines, and valproic acid which have a broad spectrum of anticonvulsant efficacy act selectively on GABA receptors; and the succinimides and oxazolidinediones which are primarily effective against petit mal seizures do not act on either of these neural substrates. The proposed research will proceed in 3 phases. (1) The binding and action of anticonvulsant drugs at sodium channels and GABA receptors in synaptosomal fractions from mammalian brain will be studied using specific radioligand binding and ion flux assays. Values of KD for drug binding and EC50 for drug action will be derived for representatives of each class of anticonvulsants. Quantitative correlations of these data with therapeutic brain levels of anticonvulsants will be made in rat and monkey brain to rigorously test the hypothesis that some classes of anticonvulsants act selectively at either sodium channels or GABA receptors at pharmacologically relevant concentrations.. (2) For those anticonvulsants which act selectively on sodium channels in mammalian brain the mechanism of drug action will be analyzed in detail using speciffic neurotoxin binding and ion flux assays in synaptosomes. (3) We have recently developed methods to purify protein components of sodium channels from rat brain and to reconstitute sodium channel function from detergent solubilized preparations. We will use these techniques to examine the molecular mechanism of anticonvulsant action on purified sodium channels. Anticonvulsant receptor site() on protein subunit(s) of sodium channels will be identified and characterized. The role of membrane phospholipid in anticonvulsant action will be assessed. These studies will provide a rational basis for the specific pharmacological profiled of anticonvulsant action and will develop the first information on the molecular basis of anticonvulsant action on purified sodium channel preparations.

membrane permeability; ion transport; calcium;

[unreadable] DESCRIPTION (provided by applicant): The threshold for action potential generation and the frequency of firing of central neurons depend critically on the cell surface density, localization, and functional properties of Na channels. Control of the cell surface density and localization of Na channels is a critical aspect of neuronal function. Previous results suggest that assembly, cell surface insertion, and differential targeting of Na channel subtypes all play important roles in this process. Cloning and functional analysis of the Beta1 and Beta2 subunits of brain Na channels have implicated these two proteins in modulation of Na channel gating, assembly of functional channels, and expression and localization of Na channels on the cell surface. In addition, the immunoglobulinlike folds in the extracellular domains of the Beta subunits suggest that they function as cell adhesion molecules. In our recent research, we have defined the new structural requirements for modulation of gating by the Beta1 subunit, discovered novel protein-protein interactions of Beta subunits with the extracellular matrix protein tenascin, the cell adhesion molecule neurofascin, and the transmembrane receptor phosphoprotein tyrosine phosphatase Beta, and analyzed the functional roles of Beta subunits in vivo in mice with disrupted 13 subunit genes. In the next project period, we plan to identify the molecular determinants for functional interaction of Na channel a and Beta subunits, investigate the molecular determinants for interaction of Na channel Beta subunits with cell adhesion and extracellular matrix molecules, define the molecular basis for regulation of Na channels by associated protein tyrosine kinases and phosphoprotein tyrosine phosphatases., identify novel molecular components of Na channel signaling complexes and determine their functional role, and examine the functional role of Na channel Beta subunits and signaling complexes in vivo in genetically altered mice. Our results will add substantially to understanding of the cell biology of the Na channel and the functional significance of Na channel signaling complexes.

Voltage-sensitive calcium channels mediate calcium influx and calcium-dependent depolarization in response to changes of membrane potential in many excitable tissues. In the heart, calcium influx mediated by these channels is responsible for the plateau phase of the cardiac action potential and initiates excitation-contraction coupling. The positive inotropic and chronotropic of norepinephrine and other Beta-adrenergic agents mediated by cAMP-dependent protein phosphorylation is due in part to an increase in the number of calcium channels activated during the cardiac action potential. In this research, radiolabeled organic calcium antagonists such as [H3]-nitrendipine will be used as probes to monitor purification of the protein components of the calcium antagonist receptor of the voltage-sensitive calcium channel. The calcium antagonist receptor will be solubilized with digitonin and purified by conventional and affinity chromatographic procedures. Preliminary results show that the purified calcium antagonist receptor consists of a complex of three subunits. The purified receptor will be characterized with respect to subunit size, composition, stoichiometry, and general biochemical properties. The purified calcium antagonist receptor will be incorporated into phospholipid vesicles and its functional activity in ion transport will be assessed by isotopic flux measurements to determine whether the purified receptor contains all the components required to mediate voltage-sensitive calcium conductance. The subunits will be separated under native conditions and the functional role of the individual subunits assessed by reconstitution. Sites of phosphorylation of the subunits of the calcium channel will be identified both in vitro and in vivo and the functional effects of pholphorylation will be analyzed. The structure of the calcium channel will be investigated further by preparation and molecular cloning of cDNA encoding the channel subunits. These cDNA clones will then be sequenced and the amino acid sequence and secondary structure of the calcium channel subunits will be derived from the nucleotide sequence to give a detailed structural model of the calcium channel. These results will provide a basis for analysis of the molecular basis of calcium channel function and physiological regulation.

pharmacology; peptide chemical synthesis; nucleic acid chemical synthesis; biomedical equipment purchase; molecular biology;

Functional properties of the major cell types in the cardiovascular system are regulated by a cascade of physiological and biochemical reactions beginning at the cell membrane and leading inward to the nucleus. In this Program Project, the structure, function, and regulation of cardiac forms of both sodium and potassium channels will be examined. cDNA's encoding cardiac forms of the protein components of these ion channels will be isolated and the primary structures of these proteins will be inferred from cDNA sequence. These channel proteins will be isolated from cardiac tissue and characterized. The molecular basis for their specific functional and pharmacological properties will be examined by co-expression of mRNA's encoding different combinations of subunits and by expression of mutant forms of the ion channel proteins. The regulation of these ion channels by protein phosphorylation and interaction with G proteins will also be studied by co-expression with mRNA's encoding normal and mutant forms of these regulatory proteins. Muscarinic acetylcholine receptors in cardiac cells bind acetylcholine and regulate cellular function by coupling through G proteins to adenylate cyclase, phosphatidylinositol hydrolysis, and voltage-sensitive ion channels. The molecular basis for this regulatory process will be examined by transfection of normal and mutant genes encoding specific muscarinic receptor subtypes, G proteins, and protein kinases into appropriate recipient cells and analysis of the resulting cells for receptor function and modulation. Hormonal regulation of contractile force in cardiac muscle involves activation of adenylate cyclase activity to increase intracellular levels of cAMP, activation of cAMP-dependent protein kinase, phosphorylation of the voltage-sensitive calcium channels and finally termination of the intracellular signal by hydrolysis of cAMP by cyclic nucleotide phosphodiesterases. The cDNA's encoding the cardiac form(s) of this key regulatory molecule will be isolated, their structures will be determined, and their functional properties will be examined in cellular expression experiments. A novel cGMP-inhibited form of cyclic nucleotide phosphodiesterase will be cloned and expressed and the receptor sites on this phosphodiesterase form for novel cardiotonic drugs will be studied by protein chemistry and site- directed mutagenesis. The development, function, and regulation of the cardiac cell requires the establishment and maintenance of specific functional domains. This coordinated program of investigation of cellular signal transduction in the heart will greatly enhance our understanding of the molecular basis of cardiac function and regulation.

Voltage-gated calcium channels respond to changes in membrane potential and mediate the entry of calcium into many cell types where it serves as a second messenger to initiate intracellular regulatory and metabolic events. These channels are subject to complex regulation by hormones, neurotransmitters, and second messengers, and are the molecular target for several important classes of drugs. The skeletal muscle calcium channel has two physiological roles. Like calcium channels in other tissues, it mediates voltage-dependent calcium entry into the cytosol. It also serves as a voltage sensor for excitation-contraction coupling linking depolarization of the transverse tubule membrane to release of calcium from the sarcoplasmic reticulum via an unknown mechanism. The abundance of this calcium channel in skeletal muscle have made it a valuable model for studies of the molecular properties of calcium channels. In previous work, we have purified, reconstituted, determined the subunit structure, studied the regulation by protein phosphorylation, and identified 175 kDa and 212 kDa size forms of the principal alpha1 subunit of the skeletal muscle calcium channel. The studies proposed here will combine biochemical, molecular genetic, and electrophysiological approaches to define the functional properties of the individual calcium channel subunits and to establish the structure- function relationships for regulation of calcium channel ion conductance activity by post translational processing and subunit assembly and protein phosphorylation. We will define the carboxyl terminal amino acid sequence of the 175 kDa form of the alpha1 subunit and determine the localization of muscle fibers of its 212 kDa and 175 kDa forms. The ion conductance and regulatory properties of skeletal muscle calcium channels containing the 175 kDa and 212 kDa forms of the alpha1 subunit will be defined. The functional roles of the individual subunits of the calcium channel in expression of its ion conductance activity in mammalian somatic cells will be studied. Physiological sites of phosphorylation of skeletal muscle calcium channels will be identified and their role in regulation of ion conductance activity will be examined. This work will give molecular insight into the complex regulation of the ion conductance activity of this class of calcium channels and equally importantly, will provide a molecular basis for understanding the function and regulation of the L- type calcium channels which are expressed in much lower densities in neurons, cardiac cells.

Ca channels transduce the cell surface electrical signals in neurons and other excitable cells into intracelular regulatory processes which control contraction, secretion, neurotransmission and gene expression. Studies of neuronal Ca channels in our laboratory and others show that the N-type and L-type channels have distinct subcellular localizations and serve distinct functions in neuronal signal transduction. These channel types are generally similar in their voltage-dependent activation, deactivation, and ion selectivity, but they have physiologically significant differences in their rates and mechanisms of inactivation, their modulation by protein phosphorylation and G proteins, and their interactions with effector proteins including synaptic membrane proteins and cellular signaling proteins. Inactivation, modulation, and interaction with cellular effectors and signaling proteins are likely to be interactive processes, and all three are likely to be determined substantially by protein-protein interactions at the intracellular surface of the Ca channels. While the membrane-associated regions of the alpha1 subunits of neuronal Ca channels have highly homologous primary structures, the intracellular loops connecting the transmembrane domains are highly divergent. Thus, the distinct localization and function of these Ca channels likely depends primarily on the unique structures of their intracellular surface. In the experiments proposed in this application, we will determine the molecular basis for the unique function and modulation of L-type and N-type neuronal Ca channels focussing on the intracellular surface of the Ca channel protein as a primary site at which these events are initiated. Our Specific Aims are to define the sites and mechanisms of modulation of the class B N-type and class C L-type Ca channels by protein phosphorylation; to define the sites and mechanisms of modulation of class B N-type Ca channels by direct interaction with G proteins; to determine the mechanism and physiological significance of receptor-dependent proteolytic processing of the carboxyl terminal domain of the alpha1 subunit of the class C L-type Ca channel; and to identify the sites of interaction of N- type Ca channels with synaptic membrane and synaptic vesicle proteins, examine the physiological role of these interactions in the processes of docking and exocytosis of neurotransmitters from synaptic vesicles, explore their regulation by protein phosphorylation and G proteins; and search for other intracellular signaling proteins which interact with Ca channels. The results of these experiments will provide essential new information required to understand function of Ca channels in cellular signal transduction in neurons and other cell types.

sodium channel; potassium channel; protein structure function; phosphorylation; heart pharmacology; biological signal transduction; drug receptors; protein kinase A; protein kinase C; heart cell; second messengers; calcium flux; antiarrhythmic agent; molecular site; action potentials; gene expression; heart contraction; Xenopus oocyte; laboratory rabbit; laboratory mouse; laboratory rat; in situ hybridization; voltage /patch clamp; transfection; western blottings; immunocytochemistry; immunoprecipitation; chimeric proteins;

This proposal requests support for an inverted confocal microscope with UV laser to provide technology which is currently unavailable to and is pivotal for the research effort of ten independent laboratories in the biomedical sciences. Research programs in all ten laboratories are now at a critical phase where long term access to this equipment is essential for further progress. Eight NIH funded users and two other funded groups are identified whose research programs include the following areas of investigation: Cell division, development, structure and function of opioid receptors, long term potentiation, cyclic nucleotide phosphodiesterases, synaptogenesis and synaptic structure, membrane traffic and redistribution of organelles within a cell and the associated changes in second messengers, interactions between calmodulin and neuromodulin, a major constituent of neuronal growth cones, and the expression and localization of voltage-gated ion chamois at the neuromuscular junction and throughout the brain. The broad range of projects proposed by the initial user group is representative of a larger group of potential users of confocal microscopy. The requested instrument would be placed in the established Keck Imaging Facility located on the University of Washington and be made available to other investigators when it is not being used by the core group of researchers. The operation and management of the equipment, the internal advisory committee and institutional support for the confocal microscope are described.

Protein phosphorylation was discovered by Edwin Krebs and Edmond Fischer in the 1950s as a crucial aspect of regulation of glycogen metabolism. In the ensuing 40 years, it has become apparent that protein phosphorylation is the most ubiquitous regulatory mechanism in cell biology. It plays a central role in such diverse biological processes as cell migration and histogenesis in early development; hormone action; regulation of cardiovascular function; synaptic plasticity, learning, and memory in the nervous system; and control of cell growth and division in all cell types. Protein phosphorylation is also implicated in cellular dysfunctions which lead to disease such as altered growth regulation in cancer, failure of insulin regulation of glucose metabolism in diabetes, and altered phosphorylation of microtubules and microfilaments in Alzheimer's disease. What are the common themes that emerge from studies of protein phosphorylation in all of these varied cellular contexts? Our meeting is designed to reveal common themes by bringing together experts in the mechanisms of cellular regulation by protein phosphorylation in many different cell types for reflection on the development of the field and for intense consideration of the avalanche of recent advances in our understanding of the role of protein phosphorylation in different regulatory pathways. We believe that this broad consideration of the mechanisms through which protein phosphorylation participates in cellular regulation in endocrine pathways, in control of cell division, growth, and differentiation, and in regulation of the cardiovascular and nervous systems will be a catalyst for further research in the field and for new interdisciplinary initiatives to unravel the complexities of the phosphorylation networks which control cell function. The meeting is organized around a series of Symposia featuring lectures by leading scientists, Workshops featuring short talks on recent research, and Poster Sessions emphasizing presentations by graduate students and postdoctoral fellows. Support is requested for travel expenses of symposium speakers and workshop coordinators.

The final common pathway of electrical excitability in neurons is generation of conducted action potentials. Action potentials in nerve and muscle are initiated by activation of voltage-gated sodium channels, and the threshold and frequency of firing which encode information in the nervous system are critically dependent on sodium channel properties. The ion conductance activity of sodium channels is controlled on the millisecond time scale by two distinct but coupled gating processes: activation and inactivation. Activation controls the voltage- and time-dependence of conductance increase in response to depolarization, and inactivation controls the voltage- and time- dependence of the subsequent return of the sodium conductance to the basal level within one millisecond. Both processes are essential for normal electrical excitability of nerve and muscle cells, and elucidation of their molecular basis is a major challenge for molecular neurobiology. The essential nature of the inactivation process is illustrated by the striking effects of dominant mutations which impair this process in the periodic paralyses of skeletal muscle and long QT syndrome in the heart. One can anticipate that similar hyperexcitability syndromes may be caused by mutations in brain sodium channels and contribute to both inherited and spontaneously arising forms of epilepsy. In the current project period, we have made substantial progress on several topics related to the molecular basis for sodium channel inactivation, its modulation by second messenger- activated protein phosphorylation and by peptide neurotoxins, and its interaction with pore-blocking drugs. We have discovered the key amino acid residues in the inactivation gate which are essential for its function, elucidated the three-dimensional structure of the key region of the inactivation gate, identified candidate residues involved in formation of the inactivation gate receptor, defined the phosphorylation sites responsible for regulation of channel gating by protein phosphorylation initiated by the dopamine D1/cAMP-dependent protein kinase signaling pathway and the muscarinic acetylcholine/protein kinase C signaling pathway in neurons, and identified important components of the receptor site for scorpion toxins and local anesthetic drugs which interact with the inactivation process. In the next project period, we propose to build on this foundation of molecular information about sodium channel gating and further define the molecular basis of its physiological and pharmacological regulation. Our objectives are to elucidate the molecular interactions of the inactivation gate with the putative inactivation gate receptor, to define the three-dimensional structure of the inactivation gate, to probe the molecular mechanisms by which protein phosphorylation influences sodium channel gating and define the interactions of the relevant phosphorylation sites with the inactivation gate, to determine the molecular basis for high affinity binding of local anesthetics to inactivated channels and for molecular trapping of these drugs in their receptor site by closure of the channel activation and inactivation gates, and to analyze the coupling of movements of the S4 voltage sensors to voltage-dependent activation and inactivation using alpha- and beta-scorpion toxins as specific molecular probes. These proposed studies will give new insight into the molecular mechanisms of sodium channel gating and its modification by second messenger-activated protein phosphorylation and by drugs and neurotoxins.

L-TYPE Ca channels containing Cav1.2 alpha1 subunits are responsible for Ca entry that initiates contraction in cardiac and smooth muscle, and they are therefore the final common pathway for regulation of contractile force by many different effectors such as neurotransmitters, hor5moines and drugs. Because of they key role in regulation of contraction, many different intracellular second messengers converge on these Ca channels and regulate their function, including cAMP, Ca, calmodulin, diacylglycerol, and ATP. Recent studies show that the sites of action of many of in these intracellular second messengers are in the large intracellular C-terminal domain, which is comprised of more than 660 amino acid residues representing 30% of the mass of the alpha1subunit, and in the smaller interacting intracellular N-terminal domain. In addition, the C-terminal domain is subject to regulated proteolysis, which modulates its function. Thus, the N-terminal and C-terminal domains interact with each other and integrate many kinds of cellular regulatory signals, which together comprise an intracellular signaling network controlling Ca channel activity. We propose to analyze the functional interactions among the regulatory sites for cAMP-dependent protein kinase, protein kinase C, A kinase anchoring protein 15 (AKAP-15) and ATP in the N-terminal and distal C-terminal domains of Cav1.2 Ca channels, to examine the cellular mechanism and functional significance of regulated proteolysis of the C-terminal domain of Cav1.2 channels, to define the molecular basis for interaction of Ca and calmodulin with the C-terminal domain of Cav1.2 channels, to define the molecular basis for interaction of Ca and calmodulin with the C-terminal domain and their regulation of Ca channel function, and to determine the structural basis for functional interactions among multiple regulatory pathways impinging on the C-terminus of Cav1.2 channels. Our experiments will use a combination of structural, biochemical, cell biological, electrophysiological, and mouse genetic approaches to reveal the molecular basis for Ca channel regulation and its role in cardiovascular physiology. The results will shed new light on the mechanisms of regulation of cardiac beating rate and contractility through an intracellular signaling network impinging on the L-type Ca channels.

[unreadable] DESCRIPTION (provided by applicant): Voltage-gated Na channels are responsible for initiation and propagation of the action potential in vertebrate nerve and muscle. Because of its essential physiological role in movement, the Na channel is a prime target of paralytic neurotoxins, which act at five or more distinct neurotoxin receptor sites. The genes encoding the polypeptide scorpion toxins have been cloned and successfully expressed in bacteria to produce large amounts of these toxins. Therefore, these toxins constitute a substantial terrorist threat as peptides. Moreover, bacteria or viruses expressing the potent polypeptide scorpion toxins are themselves terrorist threats because infection of human hosts with these agents would result in paralysis. The central hypothesis of the work proposed here is that toxin antagonists can be produced that will protect broadly and effectively against paralytic peptide neurotoxins. This hypothesis is supported by a proof-of-concept from our current research, in which the first antagonist of scorpion toxin action has been produced. In the research proposed here, we will define the receptor sites and mechanisms of action of the a- and [unreadable]-scorpion toxins on Na channels, and we will develop therapeutic agents to prevent their toxic actions as well as the toxic actions of mechanistically related peptide neurotoxins from other sources. Our Specific Aims are: 1. Molecular mapping of the scorpion toxin receptor sites on Na channels. 2. Molecular mapping of the active sites of a- and [unreadable]-scorpion toxins. 3. Three-dimensional models of the scorpion toxin receptor sites. 4. Development of novel and potent toxin and small peptide antagonists. All of our work in Specific Aims 1 through 3 immediately flow into the design and development of toxin antagonists in Specific Aim 4 and will significantly advance the effort to develop novel therapeutic agents to protect against the threat of paralytic neurotoxins. These studies will provide new insights into the molecular mechanisms of toxin action on Na channels and will lead to development of effective antagonists of toxin action. These advances will be of crucial importance to developing an arsenal of counter-terrorism agents to prevent illness and deaths from potential bioterrorist attacks using these potent paralytic neurotoxins. In addition to these important advances for counter-terrorism, these studies will shed new light on the molecular mechanisms of voltage sensing and activation gating of Na channels, an essential step toward understanding the molecular mechanisms of electrical excitability and potentially a novel approach to development of drugs to treat chronic pain and neurological disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): L-type Ca currents conducted by Cav1.2 channels are responsible for Ca entry that initiates contraction in cardiac muscle, and these channels are therefore the final common pathway for regulation of Ca signaling and contractile force by many different effectors such as neurotransmitters, hormones and drugs, and their receptors. Alterations in L-type Ca currents are crucially involved in cardiovascular disease and therapy. Misregulation of L-type Ca currents contributes to hypertension, and Ca channel antagonist drugs are an important mode of therapy. Ischemic heart disease is often accompanied by angina pectoris, which is also treated with Ca antagonist drugs. Arrhythmias can be generated by altered regulation of L-type Ca currents and by inappropriately timed Ca transients generating early and delayed afterdepolarizations, and Ca antagonist drugs are important in treatment of atrial arrhythmias, (-adrenergic regulation of L-type Ca currents is altered in heart failure. Surprisingly, despite their importance in cardiovascular physiology and pathophysiology, regulation of Cav1.2 channels in cardiac myocytes is not well understood. Because of their key role in regulation of contraction, many intracellular regulators and second messengers converge on these Ca channels and regulate their function, including Mg, cAMP, Ca, and calmodulin. Our work has shown that the sites of action of these second messengers are in the large intracellular C-terminal domain, which represents approximately 30% of the mass of the al subunit. In addition, the C-terminal domain is subject to proteolytic processing, which modulates its function. Thus, the C-terminal domain integrates many kinds of cellular regulatory signals, which together form an integrated intracellular signaling network controlling Ca channel activity. In this project we propose to determine the molecular mechanism and physiological significance of Cav1.2 channel autoinhibition the C-terminal domain, define the mechanism and physiological significance of proteolytic processing of the C-terminal of Cav1.2 channels, and determine the molecular mechanism of regulation of the Cav1.2 channel by the (-adrenergic receptor pathway acting through PKA bound to the channel's distal C-terminal domain by AKAP15. The results of our experiments will be crucial for understanding regulation of Ca and cAMP signaling in the cardiac myocyte and its dysfunction in cardiovascular disease. This information will provide the essential basic science background for translational research aimed at preventing and treating cardiovascular disease.

[unreadable] DESCRIPTION (provided by applicant): P/Q-type Ca currents conducted by Cav2.1 channels are responsible for the Ca entry that initiates neurotransmitter release at most fast glutamatergic synapses. Ca entering through presynaptic Ca channels forms a local domain of high Ca concentration that activates exocytosis in the near vicinity. Therefore, synaptic vesicles must dock near presynaptic Ca channels to be efficiently released. Neurotransmitter release is dependent on the third or fourth power of the Ca current through the presynaptic Ca channels, so small changes in Ca entry have large effects on synaptic transmission. Ca-dependent facilitation and depression of synaptic transmission is an important determinant of information coding and transmission in the nervous system. Our results in the present project period have given important new insights into the function and regulation of presynaptic Ca channels in synaptic transmission and short-term synaptic plasticity. First, we have further defined the molecular mechanism for interaction of Ca channels with SNARE proteins and the regulation of that interaction by protein phosphorylation. Second, we have shown that the calmodulin-like neuronal Ca sensor (nCaS) protein VILIP-2 regulates Cav2.1 channels by interaction at the same binding site as calmodulin and CaBP1, but has a distinct set of regulatory effects. Third, we have found that N-terminal myristoylation is required for the distinct regulatory effects of CaBP1 and VILIP-2 on Cav2.1 channels and that the N-terminal lobe of these nCaS proteins confers their specificity of regulation. Fourth, we have discovered that nCaS-dependent facilitation and inactivation of Cav2.1 channels is primarily responsible for short-term facilitation and depression of synaptic transmission in transfected superior cervical ganglion (SCG) neuron synapses, providing the first insight into the molecular mechanisms responsible for short-term synaptic plasticity. Finally, we have found unexpectedly that Ca/calmodulin-dependent protein kinase II (CaMKII) regulates Cav2.1 channels by specific binding to a site on the C-terminal domain, potentially positioning the kinase for rapid response to Ca entry and phosphorylation of nearby proteins. In the next project period, we plan to build on these important advances to: 1. further define the molecular mechanisms of binding and regulation of Cav2.1 channels by nCaS proteins; 2. determine the functions of nCaS proteins in short-term synaptic plasticity; 3. explore the signaling functions of CaMKII specifically bound to Cav2.1 channels; and 4. determine the functional role of presynaptic CaMKII in synaptic transmission and synaptic plasticity. These experiments will provide novel insights into the regulation of presynaptic Ca channels and the role of this regulation in short-term synaptic plasticity, an essential form of information encoding and transmission in the nervous system. PUBLIC HEALTH RELEVANCE: Calcium channels in nerve terminals begin the process of synaptic transmission, which communicates information from one nerve to cell to another as well as to muscle and hormone-secreting cells. Failure of correct function and regulation of these calcium channels contributes to epilepsy, migraine, ataxia, and other neurological diseases. Our proposed research will provide novel insights into the regulation of these presynaptic calcium channels and their function in short-term synaptic plasticity, an essential process for normal coding and transmission of information in the nervous system and a target for neurological disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Electrical signaling is a nearly ubiquitous property of cells ranging from bacteria to vertebrate neurons. Cellular regulation in nerve, muscle, endocrine, exocrine, epithelial, and lymphatic cells depends on electrical signaling mediated by ion channels, and many diseases are caused by dysfunction or misregulation of ion channels. Electrophysiological studies were revolutionized by the invention of the patch clamp method by Neher, Sakmann and their colleagues, for which they received the Nobel Prize in Physiology or Medicine in 1991. As originally developed, this method allows the recording of electrical signals in single cells or single patches of membrane using microelectrodes. Modern electrophysiology is entirely dependent on the patch voltage clamp method, but it is very slow and places severe restrictions on the number and type of experimental manipulations that can be performed during an experiment. Instruments for automated patch clamp recording have been in development for several years, and now have reached the high level of sophistication, technical capability, and flexibility that are required for cutting-edge research on ion channels. We are requesting a Nanion Patchliner NPC-16 automated patch clamp system, which is capable of routine extracellular and intracellular microfluidic perfusion of multiple single cells simultaneously, recoring from up to 48 cells in automated mode, and recording at elevated temperatures. This instrument substantially extends what can be accomplished using the standard patch clamp technique, greatly speeds the process of data collection and analysis, and greatly reduces reagent costs. This new instrument will enhance the basic research efforts of a group of leading ion channel researchers and will contribute to further understanding of ion channel function in health and disease.

ABSTRACT Voltage-gated sodium (Nav) channels initiate action potentials in the heart, and voltage-gated calcium (Cav) channels initiate excitation-contraction coupling. They are related proteins with a common evolutionary ancestor, and they are molecular targets for Class I and Class IV antiarrhythmic drugs (AADs) used in control of life-threatening cardiac arrhythmias. The structural basis for AAD action is unknown. We have determined the crystal structure of an ancestral bacterial Nav channel (NavAb) at 2.7 Å resolution and revealed the structural basis for voltage sensing, pore opening and closing, ion selectivity, and slow inactivation. This structure also revealed fenestrations that lead laterally from the lipid bilayer into the pore and provide an access pathway for entry of pore-blocking AADs. We constructed a Ca-selective form of NavAb, termed CavAb, and used this construct to reveal the structural basis for Ca selectivity at atomic resolution. We are now focusing on the structural basis for state-dependent block of Nav and Cav channels by AADs. CavAb is blocked by all three structural subclasses of Class IV AADs in a state-dependent manner with nM affinity. We found that the phenylalkylamine verapamil binds to a receptor site in the pore, at the inner end of the ion selectivity filter, and physically blocks it. In contrast, amlodipine and other dihydropyridines bind at a site on the lipid-facing outer surface of the pore module, at the interface between two voltage-sensing modules, and allosterically block the pore. These results reveal drug-receptor complexes of Cav channels for the first time and set the stage for complete analysis of the mechanism of state-dependent block of Nav and Cav channels at the atomic level. Our proposed experiments have three goals. 1. We will build upon strong preliminary data to reveal the high-resolution structure of the therapeutically important benzothiazepine diltiazem bound to its receptor site in the pore of CavAb, compare the chemistry of its binding to verapamil, determine the role of fenestrations in state-dependent block of CavAb, and explore the effects of mutations that substitute human residues in the AAD receptor site. 2. We will build on strong preliminary data to reveal the high- resolution structures of Class 1 AADs such as lidocaine and flecainide bound to NavAb, differentiate among the binding poses and receptor site conformations for Subclass 1A, 1B, and 1C AADs, determine the role of fenestrations in state- dependent block of NavAb, and explore the effects of mutations that humanize the NavAb drug receptor. 3. Based on a new homogeneous biochemical preparation, we will use cryo-electron microscopy and X-ray crystallography to determine the structure of a mammalian cardiac Nav1.5 channel at high resolution, define the structural basis for its unique physiological properties, and elucidate the structural basis for AAD block of Nav1.5 channels. Our results will be crucial for understanding and improving therapy of life-threatening cardiac arrhythmias by AADs.

DESCRIPTION (provided by applicant): Voltage-gated calcium (Ca) channels initiate excitation-contraction coupling in cardiac myocytes. Stimulation of the sympathetic nervous system activates ¿-adrenergic receptors, adenylyl cyclase, and cAMP-dependent protein kinase (PKA). PKA phosphorylates Cav1.2 channels and increases their activity, which contributes to increased beating rate and contractile force in response to exercise, stress, and fear. Cav1.2 channel activity is also regulated by voltage-dependent potentiation and Ca-dependent facilitation, and phosphorylation by PKA and Ca/calmodulin-dependent protein kinase II (CaMKII) is involved in this regulation. Our recent research has revealed unexpected complexity in regulation of Cav1.2 channels by PKA. First, in acutely dissociated ventricular myocytes, an A Kinase Anchoring Protein (AKAP) is required for anchoring of PKA to the distal C- terminal domain (DCT). Second, in vivo proteolytic processing severs the C-terminus near its center, potentially separating the DCT from the Cav1.2 channel. Third, the proteolytically processed DCT binds noncovalently to the proximal C-terminal domain and inhibits Cav1.2 channel activity. This autoinhibitory signaling complex with noncovalently bound DCT, AKAP, and PKA is the primary substrate for regulation PKA, which phosphorylates the channel near the site of interaction of these two halves of the C-terminus and disinhibits channel activity. We have used proteomic methods to identify novel Ser/Thr residues that are phosphorylated in vivo in response to ¿-adrenergic receptor/PKA signaling. Phosphorylation of Thr1704 by casein kinase II is important for setting basal Cav1.2 channel activity, whereas Ser1700 is required for regulation by PKA. Mutation of these phosphorylation sites in mice prevents ¿-adrenergic regulation of Cav1.2 channels in ventricular myocytes. Moreover, mice in which the DCT is deleted have marked hypertrophy and heart failure, indicating that this autoinhibitory signaling complex is required for normal cardiovascular function in vivo. Our proposed experiments will address three aims. 1. We will use unbiased co-immunoprecipitation and proteomic methods to identify AKAPs that bind to Cav1.2 in the heart, and the functional role of these AKAPs in channel regulation will be determined. 2. We will analyze voltage-dependent potentiation and CaMKII-dependent facilitation of full-length, truncated, and truncated+DCT channels in transfected cells using mutants at the Ser1700 site to determine its functional role, and we will define the functional role of this site in vivo using S1700A mice. 3. We will examine changes in the levels of full-length, truncated, and truncated+DCT Cav1.2 channels and their interactions with AKAPs in the ¿-adrenergic hyperstimulation model of heart failure, in which our preliminary studies reveal substantial molecular remodeling of Cav2.1. We will use our S1700A mice to define the role of phosphorylation of Ser1700 in hypertrophy and heart failure in vivo. These studies will increase understanding of regulation of the heart by the sympathetic nervous system and give essential new insight into the molecular and functional changes in the Cav1.2 signaling complex in heart failure.

DESCRIPTION (provided by applicant): In the heart, Ca entry through Cav channels initiates excitation-contraction coupling, and their dysregulation is important in heart failure. Selective C entry is crucial for function of Ca channels because extracellular Na is present at 70-fold higher concentration, yet the structural basis for Ca selectivity is unknown. Building on our determination of the high-resolution structure of a common ancestor of Nav and Cav channels, NavAb, we have now determined the three-dimensional structure of a Ca-selective pore for the first time by constructing a Ca-specific ion selectivity filter in NavAb. This experimental approac will allow high-resolution analysis of structural determinants of Ca binding, selectivity, permeation, and block. Mammalian Cav channels are blocked by Ca antagonist drugs used in therapy of hypertension, angina pectoris, and cardiac arrhythmia. Phenylalkylamines, benzothiazepines, and dihydropyridines bind at three well-characterized receptor sites. Remarkably, Ca antagonist drugs also block CavAb, which therefore provides a structural template for understanding block of Ca channels by drugs that are widely used in treatment of cardiovascular disease. To define the structural basis for pore function and pharmacology of Cav channels, we will address three Specific Aims. 1. We will determine the structural basis for Ca binding and selectivity in CavAb. We will measure the permeability ratio of Ca/Na for NavAb, CavAb, and intermediate mutants. We will determine the structures of the NavAb/CavAb series of mutants in the absence and presence of Ca, identify the binding sites for Ca in the pore, and estimate the relative affinity of Ca for sites in NavAb and CavAb. The affinity for specific bindin sites in the pore will be correlated with affinity values estimated from electrophysiological studies and fitting ion conductance measurements to a biophysical model of Ca binding, selectivity, and permeation. 2. We will determine the structural basis for cation block of the CavAb pore. Large divalent and trivalent cations block mammalian Cav channels and CavAb with high affinity. We will measure the affinities of these cations for block of Ca permeation, identify their binding sites in the pore, and compare affinity for binding to specific sites identiied by x-ray crystallography with block of Ca permeation. 3. We will explore the structural basis for block of CavAb by Ca antagonist drugs. We will determine the structure of CavAb with Ca antagonist drugs bound in order to understand the molecular basis for channel inhibition at high resolution. We will examine drug binding in CavAb crystals in the presence and absence of Ca in order to understand the complex interactions between Ca binding and drug block. We will create humanized chimeras of CavAb to define the structural basis for high- affinity binding and block of human Cav channels. We will correlate observations of Ca and drug binding in our crystal structures with electrophysiological analysis of drug block of Ca conductance, and we will test biophysical models by fitting kinetics and equilibrium binding parameters to electrophysiological data. Our results will give the first high-resolution insights into Cav channe structure, function, and pharmacology.

DESCRIPTION (provided by applicant): We are requesting funds for the purchase of a Leica SP8 WLL confocal microscope. The instrument will be placed in the University of Washington's Keck Imaging Facility. The Keck Facility is a well- established, very heavily used facility providing confocal microscope instrumentation for 20 years, serving over 50 UW research laboratories in 2012. The facility now has two outdated and well-worn confocal microscopes, one of which is no longer supported by its manufacturer. The requested instrument is required to sustain microscope access for our group of 50 users and to provide expanded technical capabilities that are necessary to a group of 6 major users for advancing research projects ranging from dynamics within single cells to development of entire embryos. Thus, the proposed instrument will be required to analyze a vast diversity of specimens, in both fixed and living conditions. Significantly, the proposed instrument will make unique capabilities and badly needed additional confocal capacity available to the entire Keck user base and larger UW research community. It is to achieve these goals that we are requesting the Leica SP8 WLL confocal microscope with an inverted, DMI 6000 microscope stand. The system is specified with a white light laser (WLL) providing selection of any excitation wavelengths (up to 8 simultaneously) from 470 - 670 nm. Combined with Leica's extremely flexible spectral detection, fluorescent imaging parameters are user-customizable to a degree not currently available at the University of Washington. The system includes two hybrid (HyD) detectors for fluorescence. These new-generation detectors provide significantly increased sensitivity, wide dynamic range and low noise characteristics compared to standard detectors. A resonance scanner provides rapid imaging necessary for capturing dynamic events. A motorized stage and adaptive focus correction module are included for automated sampling and maintaining correct focus during time-lapse studies, respectively. A temperature-controlled stage insert is included for live-cell experiments. The Leica SP8 WLL was evaluated by the Keck Facility staff and major users at a recent demonstration and found to meet and exceed all of our expectations. In summary, the proposed instrument will permit users to optimize confocal imaging parameters, in ways currently impossible, to overcome the specimen's limitations rather than forcing experimental compromises necessary to overcome current instrument limitations.

Life-threatening pediatric epilepsies like Dravet Syndrome (DS) are unresponsive to standard therapy. We will assess the efficacy of the Nav1.6-specific sodium channel blocker XEN-A in Scn1a+/- mice, a validated mouse genetic model of this devastating childhood epilepsy disorder. DS is caused by dominant, heterozygous loss- of-function mutations in the gene SCN1A, encoding brain sodium channel Nav1.1. Our mouse genetic model exhibits all facets of DS, including thermally induced seizures, spontaneous seizures, premature death, hyperactivity, severe cognitive impairment, and autistic-like behaviors. These manifestations of DS are caused by selectively impaired action potential firing in GABAergic inhibitory neurons in hippocampus and cerebral cortex, which alters the balance of excitation to inhibition in neural circuits in favor of excitation. DS mice provide a unique resource to study the effects of XEN-A on a naturally occurring intractable epilepsy. Nav1.6 is the primary sodium channel in excitatory neurons, where it drives repetitive firing. It is a prime target for next- generation antiepileptic drugs, but none have been tested in DS. As proof-of-principle, we made DS mice that were heterozygous for a loss-of-function mutation in Nav1.6. These mice were much less susceptible to seizures and premature death, supporting inhibition of Nav1.6 as a therapeutic strategy in DS. Drug discovery research has yielded next-generation Nav1.6 inhibitors that are highly selective among sodium channel types, in sharp contrast to the nonselectivity of traditional sodium channel blockers that are not effective in DS. We will test the efficacy of a next-generation Nav1.6 drug (XEN-A, Xenon Pharmaceuticals) in our mouse model of DS. We hypothesize that Nav1.6-selective inhibitors will have potent antiepileptic effects in DS mice by specifically inhibiting Nav1.6 in excitatory neurons in a voltage-dependent manner and will further have beneficial effects on hyperactivity, cognitive deficit, and autistic-like behavior by re-balancing excitation and inhibition. We will test XEN-A on thermally induced seizures, frequency and severity of spontaneous seizures and premature death, and cognitive and behavioral deficits, including hyperactivity, repetitive behaviors, impaired spatial learning and memory, and autistic-like behaviors. We will analyze seizure mechanisms in DS mice treated with XEN-A by electroencephalographic recording (EEG) and determine mechanism of action of XEN-A in electrophysiological studies of neurons in brain slices. Our results could transform treatment of pediatric epilepsies by identifying physiological mechanism(s) that render these seizures tractable and so permit development of improved therapeutic strategies for control of intractable seizures. Determination of the underlying mechanisms will provide a solid basis for the development of a new class of antiepileptic drugs to benefit pediatric epilepsies broadly and may help to better understand key physiological processes that can be targeted to control intractable seizures.

Voltage-gated sodium (Nav) and calcium (Cav) channels generate action potentials and initiate synaptic transmission in neurons. Mutations in them cause inherited epilepsy, migraine, chronic pain, and periodic paralysis, and they are important molecular targets for drugs. A. New insights into structure and function of Nav channels have come from our high-resolution x-ray crystallography of their bacterial ancestor NavAb. We will further define the structural basis for key functional properties of mammalian Nav channels by building their characteristic structural features into NavAb, including the structural basis for voltage-dependent activation, ion selectivity, and fast inactivation. Based on these results, we will determine the structural basis for impaired Nav channel function by mutations that cause periodic paralysis and the chronic pain syndromes erythromelalgia and paroxysmal extreme pain disorder. B. Failure of learning and memory is a debilitating aspect of aging and neurodegenerative disease, yet we do not understand the basic mechanisms of these crucial brain processes and we cannot intervene effectively in these deficits. Learning and memory takes place primarily at synapses. Presynaptic calcium (Cav2.1) channels initiate neurotransmitter release at most synapses in the brain. The activity of these channels is tightly regulated by a large complex of signaling proteins, including calmodulin and related calcium-sensor proteins. Our work implicates Cav2.1 channel regulation in short-term synaptic plasticity in transfected synapses in cultured neurons and in a novel mouse model in which the IM-AA mutation is inserted into Cav2.1. We will further define the molecular and structural mechanism for Cav2.1 channel regulation, determine the role of regulation of Cav2.1 channels in short-term synaptic plasticity of neural circuits, and explore the role of regulation of Cav2.1 channels and short-term synaptic plasticity in spatial learning and memory. Our experiments with this unique mouse model will give unique insights into the mechanism of short-term presynaptic plasticity in hippocampal neurons and its role in integrative bbrain function. C. Dravet Syndrome (DS) is a devastating childhood neuropsychiatric disorder caused by de novo, heterozygous loss-of-function mutations in Nav1.1. We developed a mouse genetic model with all the features of DS, including thermally induced and spontaneous seizures, ataxia, circadian rhythm and sleep disorders, cognitive deficit, autistic-like features, and premature death via SUDEP. Physiological and genetic studies show that all these effects are correlated with loss of Na currents and excitability of GABAergic interneurons, without consistent effects on excitatory neurons, which causes imbalance of excitation vs. inhibition in neural circuits. To further advance understanding of pathophysiology and treatment of DS, we will determine the neural cells and circuits responsible for DS using specific deletion by the Cre-Lox method, identify the sites of hyperexcitability in neural cells and circuits that appear first in DS mice in vivo, and optimize next-generation combination therapy for seizures, status epilepticus, cognitive deficit, and premature death in DS.