John P. Adelman - US grants

Motilin is a gastrointestinal hormone which may regulate intestinal motility. The precise molecular nature and tissue distribution of motilin, and mechanisms of motilin induced gut contractions are controversial due to use of antisera against porcine motilin and synthetic porcine motilin for studies in non- porcine species. Paucity of anatomical and physiological information on enteric nerves in the species studied is also a compounding problem. This study focuses on the synthesis, localization and function of the prepromotilin derived peptides, motilin and motilin associated peptide (MAP). Motilin cDNA from guinea pig gut will be used to detect prepromotilin mRNA by Northern analysis and in situ hybridization. Nucleotide sequence determination will permit synthesis of guinea pig motilin and MAP for use in studies on single enteric neurons in vitro and intestinal motility studies in vivo, as well for antisera generation to study motilin/MAP processing by RIA, HPLC and immunohistochemistry. Conducting these studies in guinea pigs, a species with a well characterized enteric nervous system, will make it possible to assign motilin/MAP actions to specific neuronal sub-populations with defined interactions and effector targets. Studying the biology of motilin/MAP from the level of gene expression to physiological action(s) in the intact animal should define their role in gastrointestinal function.

The function of the Gonadotropin Releasing Hormone (GnRH) gene is central to the reproductive capability of mammalian organisms. At the cellular level many factors may act to regulate the production of the GnRH decapeptide via effects on transcription, translation and post-translational events. The structures of the mRNA and genomic locus encoding the GnRH precursor in the rat and human have been described. Further work revealed that the DNA in the rat which encodes the GnRH precursor on one DNA strand, also encodes a distinct gene, SH, on the opposite DNA strand. The SH and GnRH genes share significance exonic sequences and the RNAs transcribed from either strand show distinct but overlapping patterns of tissue and cellular specificity. The major goal of the research proposed here is to more thoroughly characterize the GnRH:SH gene locus and examine the functional relationship between the GnRH and SH gene products. First, will be to localize the promoter elements present on both strands of the GnRH:SH gene by mapping transcriptional initiation sites, and by assaying the ability of implicated genomic sequences to drive expression of reporter genes. Second, will be to determine if the SH RNAs produce proteins in vivo. Third, will be to determine whether there are cells which co-express the GnRH and SH genes. Fourth, will be to examine the consequences of expressing the GnRH and SH genes within the same cells. By studying the structure and function of the GnRH:SH gene locus in this way, we hope to answer questions regarding the regulation of the reproductive control molecule GnRH, the function of the SH gene, and the significance of this novel eukaryotic genetic arrangement.

The functions of potassium channels are central to the properties of all excitable mammalian cells. Electrophysiological and pharmacokinetic studies have defined many different classes of potassium channels. However, the molecular basis of this diversity, and the relationships between channel structure and function remain unknown. Through the use of molecular cloning and electrophysiology, we have isolated, characterized, and expressed in frog oocytes a mammalian potassium channel from rat hippocampus. Surprisingly, the properties of this channel do not fit any single kind of channel as defined by classical methods. The major goal of the research proposed here is to further our understanding of the molecular basis of variations among the different classes of mammalian potassium channels. First, we will clone, characterize, and express in frog oocytes mammalian potassium channels, both voltage and ligand gated, with distinct structural and functional properties. The different kinds of channels will be compared to those known by classical methods. Second, structure-function studies, using site directed mutagenesis, will be performed using a range of potassium channels with distinct properties. Site directed mutant studies of whole, reconstructed channels will be conducted in oocytes. Third, ancillary factors which may influence channel functions will be investigated. Fourth, subunit-specific probes will be generated, based on established nucleotide sequences, and employed in in situ hybridization studies to map the expression patterns of potassium channels in the brain. By studying the structure and function of mammalian potassium channels in this way, we hope to answer questions regarding the molecular basis of potassium channel diversity and the significance of this diversity to the basic properties of excitable mammalian cells.

The functions of potassium channels are central to the properties of all excitable mammalian cells. Electrophysiological and pharmacokinetic studies have defined many different classes of potassium channels. However, the molecular basis of this diversity, and the relationships between channel structure and function remain unknown. Through the use of molecular cloning and electrophysiology, we have isolated, characterized, and expressed in frog oocytes a mammalian potassium channel from rat hippocampus. Surprisingly, the properties of this channel do not fit any single kind of channel as defined by classical methods. The major goal of the research proposed here is to further our understanding of the molecular basis of variations among the different classes of mammalian potassium channels. First, we will clone, characterize, and express in frog oocytes mammalian potassium channels, both voltage and ligand gated, with distinct structural and functional properties. The different kinds of channels will be compared to those known by classical methods. Second, structure-function studies, using site directed mutagenesis, will be performed using a range of potassium channels with distinct properties. Site directed mutant studies of whole, reconstructed channels will be conducted in oocytes. Third, ancillary factors which may influence channel functions will be investigated. Fourth, subunit-specific probes will be generated, based on established nucleotide sequences, and employed in in situ hybridization studies to map the expression patterns of potassium channels in the brain. By studying the structure and function of mammalian potassium channels in this way, we hope to answer questions regarding the molecular basis of potassium channel diversity and the significance of this diversity to the basic properties of excitable mammalian cells.

DESCRIPTION (Adapted from the investigator's abstract): Large conductance, calcium-activated potassium channels (BK) are widely distributed and fundamentally important to excitable and secretory cells. These channels comprise a large and diverse class which respond to a combination of voltage and calcium, and possess a large unit conductance (130-300 pS). Through sensitivity to calcium, a major intracellular messenger, calcium-activated potassium channels serve to link basic cellular metabolism to membrane potential. BK channels are present in neurons where they contribute to the repolarizing phase of the action potential, and possibly to transmitter secretion. They are also found in striated and smooth muscle, endocrine and exocrine gland cells, kidney tubules and epithelia. BK channel activity is absolutely dependent upon intracellular calcium, but can be modulated by G-proteins and phosphorylation. These investigators have cloned and expressed a large family of BK-like calcium-activated potassium channels from Drosophila. Although they share an overall architecture with other cloned potassium channels, they differ significantly in primary sequence and many fundamental functional aspects. They propose to use an integrated combination of electrophysiology, molecular biology, and biochemistry to: 1. Determine the mechanisms by which calcium effects channel gating. 2. Determine the molecular basis of the large unit conductance of these channels, and 3. Determine the structure and function of mammalian calcium-activated potassium channels.

ATP is a ubiquitous intracellular metabolite which serves both as a store for chemical energy and as a second messenger. A class of potassium channels which are inhibited by intracellular ATP has been detected in a variety of tissues. K-ATP channels have been implicated in diverse metabolic processes as well as pathological conditions such as diabetes, ischemia and hypoxia, and epilepsy. A PCR-based cloning strategy has identified many novel potassium channel clones; expression of one clone isolated from rat heart resulted in functional K-ATP channels. The expressed channels show inward rectification, are inhibited by intracellular ATP or AMP-PNP, are reactivated by nucleotide diphosphates following rundown, and are sensitive to potassium channel openers such as pinacidil. In contrast to native K-ATP channels, the cloned channels are not affected by sulfonylureas such as glyburide. A highly related, but distinct clone has also been isolated from pancreatic islet cells. To understand the mechanisms which regulate K-ATP channels, we will: 1. Determine the domains and specific residues which comprise the nucleotide binding sites of cloned K-ATP channels. 2. Determine the relationship between sulfonylurea receptors and K-ATP channels. 3. Determine whether cloned K-ATP channels are sensitive to modulation by G-proteins.

DESCRIPTION: Small conductance calcium-activated potassium channels (SK channels) serve a fundamental role in excitable cells. Activated by elevated levels of intracellular calcium, SK channels mediate the slow after hyperpolarization, the sAHP, which follows the action potential spike. During a sustained stimulus, a train of action potentials is elicited and the depth and extent of the sAHP are increased with each action potential such that the cell is ultimately unable to fire a subsequent action potential even though the stimulus to fire remains. This phenomenon is termed 'spike-frequency adaptation' and protects the cell from tetanic stimulation and associated cell toxicity. Subtypes of SK channels may be distinguished by different sensitivities to the bee venom peptide toxin apamin. Application of apamin to regions of the brain alters physiologically important processes, such as sleep patterns and learning and memory. While the sAHP in most neurons is apamin-sensitive, in some neurons such as hippocampal pyramidal cells, the sAHP is apamin-insensitive, shows a slower time course, and is modulated by activation of protein kinase A (PKA).

DESCRIPTION: Skeletal muscle excitation is normally controlled by the influence of innervating nerve. However, prior to innervation, upon denervation, in patients with myotonic muscular dystrophy (DM), or myotubes cultured in the absence of nerve, skeletal muscle is hyperexcitable, in that a train of action potentials is often induced following an evoked contraction. The cellular hallmark of these conditions is the appearance of receptors for the peptide toxin apamin, a potent blocker of small conductance calcium-activated potassium (SK) channels. Indeed, application of apamin to denervated or myotonic dystrophic skeletal muscle dramatically repress the hyperexcitability, demonstrating that SK channels are central to the hyperexcitable state. We have cloned the apamin sensitive SK channels from skeletal muscle, SK3, and found that upon denervation or after differentiation of the muscle cell line, L6, the SK3 gene is expressed while in normally innervated muscle it is not expressed. Neither the physiological role of SK channels in hyperexcitable skeletal muscle nor the molecular cues controlling SK3 gene expression are yet understood. In this proposal, we will test the hypothesis that: (1). SK3 channels reside in the transverse tubules of denervated skeletal muscle cells. Patch clamp measurements will be performed using denervated normal and detubulated cultured myotubes. Immunohistochemistry using SK3 channel-specific antibodies, and I125-apamin binding studies will be performed. (2). SK channel activity induces hyperexcitability. Skeletal muscle myotubes and nerve cells will be co-cultured. SK channels will be heterologously expressed by infection with recombinant retroviruses and the cells electrophysiologically assayed. (3). The SK3 promotor is activated following differentiation of cultured L6 myoblasts. a) SK3 promotor/luciferase constructs will be introduced into L6 myoblasts, and luciferase activity assessed before and after differentiation; b) gel-shift and footprint assays will be performed with nuclear extracts from pre- and post-differentiated L6 cells; c) previously uncharacterized sequences in the SK3 promotor shown to be necessary for activation following myoblast differentiation will be used to screen a differentiated L6 skeletal muscle cDNA expression library. (4). DMAHP (myotonic dystrophy associated homeodomain protein or DMPK (myotonic dystrophy protein kinase) regulates SK channel expression. a) DMAHP and/or DMPK will be ectopically expressed in L6 myoblasts and SK3 mRNA and channel activity assessed before and after differentiation. b) gel-shift assays and footprints will be performed with the SK gene promotor and recombinant DMAHP; c) SK3 promoter/luciferase constructs will be introduced with or without DMAHP and/or DMPK into L6 myoblasts and the promoter elements responsible for regulation will be determined. These studies will establish a framework for understanding the molecular, cellular and physiological abnormalities of hyperexcitable skeletal muscle as well as the coordinate regulation of SK gene expression in muscle tissue.

DESCRIPTION: (Applicant's Abstract) Small conductance Ca2+-activated potassium channels (SK channels) are voltage-independent and activated by submicromolar concentrations of Ca2+. SK channel activity underlies the prolonged afterhyperpolarization (AHP) which follows an action potential. In CA1 hippocampal pyramidal neurons (HPNs) the AHP has two kinetic components. The medium AHP is apamin-sensitive and is not affected by neurotransmitter-induced second messengers, while the slow AHP is insensitive to apamin, and is modulated by neurotransmitter-induced second messengers that act in a convergent manner and exert strong effects on neuronal excitability. A sustained stimulus elicits a train of action potentials and with each action potential the AHP increases in depth and duration, lengthening interspike intervals until the cell is no longer able to reach action potential threshold. This phenomenon, termed spike-frequency adaptation, regulates burst frequency and is essential for normal, integrative neurotranmission. We have cloned three distinct SK channel subunits that exhibit the essential functional features of native SK channels in heterologous expression systems. The cloned SK subunits have remarkably homologous primary sequences except in their structurally divergent intracellular N- and C-terminal domains. The three SK subunits are expressed in overlapping but distinct patterns in the mammalian CNS, and all three cloned subunits are expressed in CA1 HPNs. The central hypothesis underlying this application is that the different SK subunits make distinct contributions to CA1 neuronal physiology. Specifically, a) the different SK subunits underlie the two AHP components, b) the different kinetics result from spatial and functional coupling between the medium AHP channels and voltage-gated Ca2+ channels, while the slow AHP channels are localized such that Ca2+ released from intracellular stores serves to activate them, and c) heterologous proteins associate with the SK subunits and influence subcellular distribution, regulation by second messengers, and pharmacology. First, we will construct transgenic mice through homologous recombination in which expression each of the SK subunit may be acutely regulated in vivo. We will record CA1 HPNs in brain slices and acutely dispersed cultures examining the AHP, interspike interval, spike-frequency adaptation, and coupling with L-type Ca2+ channels. Second, we will use pharmacological approaches to determine the sources of Ca2+ that activate the medium and slow AHPs in CA1 HPNs. Third, we will investigate the composition of the SK channel microdomain using the 2-hybrid system and a CA1-specific cDNA library to isolate proteins that associate with the pore-forming SK channel subunits and influence SK channel assembly, distribution, regulation by second messengers, and pharmacology.

DESCRIPTION (provided by applicant): Small conductance Ca2+-activated potassium channels (SK channels) underlie the after-hyperpolarization that follows an action potential. Within a train of action potentials, progressively longer interspike intervals are due to the medium Al-IP, while the slow AHP underlies spike-frequency adaptation whereby a burst is terminated, regulating burst frequency. A wide range of neurotransmitters increase cAMP levels, inhibiting the slow AHP, suppressing spike-frequency adaptation, and enhancing excitability. Three mammalian SK channels have been cloned, with properties consistent with the medium AHP. However, the relationship of the cloned channels to the slow AHP, and the molecular nature of the channels underlying the slow AHP remain unresolved. The larval muscle preparation from Drosophila provides a model system that has been essential for understanding the molecular physiology of K+ channels. One of the two prominent K+ currents in larval muscle that has not been molecularly identified is a slowly activating, Ca2+-activated K+ conductance, Ics that shares many of the hallmark features of the mammalian slow AHP. The Drosophila genome contains a single SK gene, expressed in embryos, larvae, and adult animals.The driving hypothesis for this application is that the Drosophila ICs is orthologous to the mammalian IsIowAHP. First, we will thoroughly characterize Ics. Second, we will knock out dSK, determine the phenotypic consequences, and test the hypothesis that dSK channels underlie Ics Third, if the dSK gene does not encode the Ics channels, we will clone the gene encoding lcs channels.

DESCRIPTION (provided by applicant): Small conductance Ca2+-activated K+ channels (SK) channels are fundamental regulators of neuronal excitability, essential for normal neurotransmission. SK channel activity shapes interspike intervals during a burst of action potential and is thought to underlie spike-frequency adaptation. Cloned SK channels share the membrane topology of the voltage-dependent K+ channel family, but are voltage-independent, being gated solely by intracellular Ca2+ ions and thus couple intracellular Ca2+ levels and membrane potential. SK channels are heteromeric complexes, consisting of a-pore-forming subunits and calmodulin (CaM), which is constitutively bound to a 76 amino acid domain of the intracellular C-terminal region adjacent to the sixth transmembrane segment, the CaM binding domain (CaMBD). Ca2+ binding to CaM initiates structural rearrangements in the CaMBD-Ca2+ -CaM complex that are transduced into conformational alterations of the channel gate and opening of the pore. Like selective permeation, gating is a critical feature of K+ channel function. However, unlike selective permeation, a detailed structural understanding of K+ channel gating has not been obtained. Towards this goal, we have recently described the crystal structure of the CaMBD-Ca2+ CaM complex resolved to 1.60 A. Surprisingly, the structure is a dimer of two CaMBD molecules complexed to two Ca2+-CaM molecules. Based upon the crystal structure and biochemical analyses suggesting that in the absence of Ca2+ the complex exists as a monomer of one CaMBD molecule and one CaM molecule, we have presented a novel model for ion channel gating in which a Ca2+-dependent dimer-of-dimers mediates chemomechanical gating. In this application we propose to obtain a complete atomic level description of the Ca2+ dependent gating mechanism of the SK channels by solving the crystal structure of the Ca+-free form of the CaMBD-CaM complex, functionally testing the dimer-of-dimers gating model, and determining the atomic details of the interactions between the SK channel gating apparatus and a drug that modulates SK channel gating, exerting profound effects on neuronal excitability. These studies will provide a framework for understanding K+ channel gating as well as the diverse mechanisms through which CaM modulates intracellular Ca2+ levels and membrane excitability. In addition, such a detailed knowledge of the gating mechanism for SK channels will be essential for rational drug design, because modulating SK channel gating may provide a therapeutic target for pathologies of neuronal hyperexcitability such as epilepsy and schizophrenia.

DESCRIPTION (provided by applicant): Small conductance Ca2+-activated K+ channels (SK) channels are fundamental regulators of neuronal excitability, essential for normal neurotransmission. SK channel activity shapes interspike intervals during a burst of action potential and is thought to underlie spike-frequency adaptation. Cloned SK channels share the membrane topology of the voltage-dependent K+ channel family, but are voltage-independent, being gated solely by intracellular Ca2+ ions and thus couple intracellular Ca2+ levels and membrane potential. SK channels are heteromeric complexes, consisting of a-pore-forming subunits and calmodulin (CaM), which is constitutively bound to a 76 amino acid domain of the intracellular C-terminal region adjacent to the sixth transmembrane segment, the CaM binding domain (CaMBD). Ca2+ binding to CaM initiates structural rearrangements in the CaMBD-Ca2+ -CaM complex that are transduced into conformational alterations of the channel gate and opening of the pore. Like selective permeation, gating is a critical feature of K+ channel function. However, unlike selective permeation, a detailed structural understanding of K+ channel gating has not been obtained. Towards this goal, we have recently described the crystal structure of the CaMBD-Ca2+ CaM complex resolved to 1.60 A. Surprisingly, the structure is a dimer of two CaMBD molecules complexed to two Ca2+-CaM molecules. Based upon the crystal structure and biochemical analyses suggesting that in the absence of Ca2+ the complex exists as a monomer of one CaMBD molecule and one CaM molecule, we have presented a novel model for ion channel gating in which a Ca2+-dependent dimer-of-dimers mediates chemomechanical gating. In this application we propose to obtain a complete atomic level description of the Ca2+ dependent gating mechanism of the SK channels by solving the crystal structure of the Ca+-free form of the CaMBD-CaM complex, functionally testing the dimer-of-dimers gating model, and determining the atomic details of the interactions between the SK channel gating apparatus and a drug that modulates SK channel gating, exerting profound effects on neuronal excitability. These studies will provide a framework for understanding K+ channel gating as well as the diverse mechanisms through which CaM modulates intracellular Ca2+ levels and membrane excitability. In addition, such a detailed knowledge of the gating mechanism for SK channels will be essential for rational drug design, because modulating SK channel gating may provide a therapeutic target for pathologies of neuronal hyperexcitability such as epilepsy and schizophrenia.

DESCRIPTION (provided by applicant): Small conductance Ca2+-activated K+ channels (SK channels) are gated directly by Ca2+ ions. In many central neurons such as CA1 hippocampal neurons, SK channel activity underlies the medium component of the after hyperpolarization (mAHP) that follows an action potential, influencing the number of action potentials and the interspike interval during a burst, thereby regulating neuronal excitability. In addition, SK channels in CA1 neurons modulate the induction of synaptic plasticity and alter hippocampal-dependent learning. Blocking SK channels with apamin reduces the stimulus intensity that is required to induce NMDA receptor-dependent long-term potentiation at Schaffer collateral synapses, and reduces the number of training trials required for hippocampal dependent learning. To determine the distinct physiological roles for each SK channel subtype, homologous recombination has been used to generate transgenic mice for each of the three SK channel genes. Animals lacking SK2 channels are hyperexcitable, and completely lack the apamin-sensitive component of the AHP. Immunocytochemistry shows that plasma membrane SK2 channels are distributed to multiple subcellular compartments, the soma, dendritic shafts where they form discrete puncta, and dendritic spines. The driving hypothesis for this proposal is that in CA 1 neurons spatially and functionally discrete SK2 channel populations, embedded in distinct Ca2+ signaling microdomains, modulate the induction of synaptic plasticity and hippocampal-dependent learning. To test this hypothesis we will first measure the induction of synaptic plasticity at Shaffer collateral CA1 synapses, and hippocampal-dependent learning in wild type and SK transgenic mice. We will determine whether SK2 channels in dendritic spines are activated by an activity-dependent rise in spine Ca2+ flowing through NMDA receptors, thereby reducing the Ca2+ current through NMDA receptors, and whether dendritic SK2 channels modulate pairing-dependent, Hebbian forms of synaptic plasticity. We will determine the physiological consequences of the interactions between SK2 channels and seven candidate interacting proteins, and isolate and characterize intact SK2 channel microdomains. These studies will employ a novel repertoire of reagents and techniques to engender an integrated understanding of the roles SK2 channels play in fundamental aspects of neuronal excitability as well as synaptic plasticity and memory encoding.

DESCRIPTION (provided by applicant): The slow afterhyperpolarization (sAHP) that follows an action potential in many central and peripheral neurons is due to the activation of Ca2+-dependent, voltage-independent K+ channels. Neurons in the CA1 region of the hippocampus have served as models for studying the sAHP and the underlying currents, the IsAHP. The results of studies performed over the past two decades show that the sAHP has a profound influence on neuronal excitability, being responsible for spike-frequency adaptation that regulates burst frequency and is essential for normal, integrative neurotransmission. In CA1 neurons, regulation of the sAHP is one of the principal targets for the ascending modulatory neurotransmitter systems that are involved in regulating the sleep-wake cycle, arousal, attention, and in modulating sensory processing, behaviors, emotions and memory consolidation. Initially, the IsAHP channels were thought to be members of the SK, small conductance Ca2+-activated K+ channel family, but recent data from SK transgenic mice consolidate and confirm the evidence that the IsAHP channels are not SK channels. Moreover, kinetic analysis of the sAHP and the IsAHP, indicate that the IsAHP channels may not be directly gated by Ca3+ ions, although elevated intracellular Ca2+ is essential for their activation. Taken together, it is now clear that the molecular identities of the sAHP channels remain unknown. We have compared the functional characteristics of cloned channels in concert with bioinformatics and cell-type expression data for all Kf channel genes to develop a priority list of 15 IsAHP channel candidates. We propose to use a combination of molecular biological and electrophysiological techniques to clone the slow AHP channel. The IsAHP channels are one of the most important and molecularly elusive channels. Determining the identities of the IsAHP channels will provide a powerful target for therapeutic approaches to multiple central and peripheral pathologies such as schizophrenia, epilepsy, attention deficit syndrome, and sleep disorders.

[unreadable] DESCRIPTION (provided by applicant): Small conductance Ca2+-activated K+ channels (SK channels) are gated directly by Ca2+ ions, via constitutively associated calmodulin (CaM). In many central neurons such as CA1 hippocampal neurons, SK channel activity underlies a medium component of the after hyperpolarization (mAHP) that follows an action potential, influencing the number of action potentials and the interspike interval during a burst of action potentials, thereby regulating neuronal excitability. In addition, SK channels in CA1 neurons modulate synaptic plasticity and alter memory encoding. Blocking SK channels reduces the stimulus intensity that is required to induce NMDA receptor-dependent long-term potentiation at Schaffer collateral synapses, and reduces the number of training trials required for hippocampal dependent learning. Therefore, modulators of SK channels will exert profound effects on integrated neuronal functions. We have found that a third protein, the serine/threonine protein kinase CK2, forms a stable and integral component of the SK2 channel complex. SK2-associated CK2 phosphorylates T80 of CaM and induces a shift in the Ca2+ sensitivity of SK2 channel gating. Additional data suggest that the N- and C-terminal domains of SK2 channels are in spatial proximity to the CaM binding domain, and all three domains interact with CK2. Further, the N-terminal domain is a strong activator of CK2, while the C-terminal domain contains numerous phosphorylation sites. The driving hypothesis for this application is that the N- and C-terminal domains of the SK2 channel regulate associated CK2 activity in response to dynamic metabolic signals and that SK2-associated CK2 activity influences neuronal excitability and the induction of synaptic plasticity. To test this hypothesis, we will identify the precise sites of interaction between SK2 and CK2 and determine the contributions of the N- and C-terminal domains to CK2 activity. We will determine high resolution structures of SK2-CaM-CK2 complexes. We will introduce CK2-independent SK2 channels into the CA1 area of SK2-null mice and determine the consequences for excitability and synaptic plasticity. These studies will employ a novel repertoire of reagents and techniques to engender an integrated understanding of multi-protein SK2 channel complexes, and their roles in fundamental aspects of neuronal excitability as well as synaptic plasticity. In addition, drugs that decrease SK2-associated CK2 activity and thereby decrease neuronal excitability may be therapeutic avenues for treatments of hyperexcitability disorders such as schizophrenia and epilepsy. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Small conductance Ca2+-activated K+ channels (SK channels) are gated directly by Ca2+ ions, via constitutively associated calmodulin (CaM). In many central neurons such as CA1 hippocampal neurons, SK channel activity underlies a medium component of the after hyperpolarization (mAHP) that follows an action potential, influencing the number of action potentials and the interspike interval during a burst of action potentials, thereby regulating neuronal excitability. In addition, SK channels in CA1 neurons modulate synaptic plasticity and alter memory encoding. Blocking SK channels reduces the stimulus intensity that is required to induce NMDA receptor-dependent long-term potentiation at Schaffer collateral synapses, and reduces the number of training trials required for hippocampal dependent learning. Therefore, modulators of SK channels will exert profound effects on integrated neuronal functions. We have found that a third protein, the serine/threonine protein kinase CK2, forms a stable and integral component of the SK2 channel complex. SK2-associated CK2 phosphorylates T80 of CaM and induces a shift in the Ca2+ sensitivity of SK2 channel gating. Additional data suggest that the N- and C-terminal domains of SK2 channels are in spatial proximity to the CaM binding domain, and all three domains interact with CK2. Further, the N-terminal domain is a strong activator of CK2, while the C-terminal domain contains numerous phosphorylation sites. The driving hypothesis for this application is that the N- and C-terminal domains of the SK2 channel regulate associated CK2 activity in response to dynamic metabolic signals and that SK2-associated CK2 activity influences neuronal excitability and the induction of synaptic plasticity. To test this hypothesis, we will identify the precise sites of interaction between SK2 and CK2 and determine the contributions of the N- and C-terminal domains to CK2 activity. We will determine high resolution structures of SK2-CaM-CK2 complexes. We will introduce CK2-independent SK2 channels into the CA1 area of SK2-null mice and determine the consequences for excitability and synaptic plasticity. These studies will employ a novel repertoire of reagents and techniques to engender an integrated understanding of multi-protein SK2 channel complexes, and their roles in fundamental aspects of neuronal excitability as well as synaptic plasticity. In addition, drugs that decrease SK2-associated CK2 activity and thereby decrease neuronal excitability may be therapeutic avenues for treatments of hyperexcitability disorders such as schizophrenia and epilepsy. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This proposal requests partial support for an internationally attended meeting on Ion Channels, as part of the Gordon Research Conference series, to be held at the Tilton School in Tilton, New Hampshire, July 6-11, 2008. The broad and long-term goal of the conference is to increase our understanding of the fundamental structure, function and physiological roles of ion channels as well as their dysfunction in disease, particularly neurological disorders. The specific aims of this meeting will be to convene 44 speakers and discussion leaders that represent critical areas of ion channel research with a total of 135 participants for a five-day meeting in a relatively secluded setting. The program will have nine sessions that broadly address current issues in ion channel regulation, biophysics, structure, physiological roles and dysfunction in disease. In addition, four poster sessions will permit all participants to contribute their work to these topics. The significance of this application is that the Ion Channels Gordon Research Conference is a critical component of the established meetings that fertilize ideas and research in the international community of ion channel biologists. Indeed, the small size and intensive discussions engendered by the Ion Channels GRC make it uniquely important for the catalysis of new ideas and directions among the participants. The health relatedness of this application is that the discussions will define questions that require experimental resolution in a wide variety of areas that affect human development and health. PUBLIC HEALTH RELEVANCE: The relevance of this application is that the detailed discussions of cutting edge research will define the questions that require experimental resolution in areas that affect human development, and excitability disorders of almost every tissue and organ system, including the central nervous system, sensory organs, the pancreas, cardiac, smooth and skeletal muscle. These questions will also be directly relevant to human aging. Ultimately, the definition and experimental resolution of these issues will pave the way for innovative therapeutic approaches to a wide variety of human diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Small conductance Ca-activated K channels (SK channels) affect excitability and contribute to shaping excitatory postsynaptic potentials (EPSPs). SK2 channels are expressed throughout the dendritic arbors of hippocampal CA1 neurons and contribute to synaptic plasticity and learning and memory. We have recently found that the SK2 gene encodes two isoforms of the SK2 protein. SK2-L contains an additional 209 N- terminal amino acids compared to SK2-S that is completely contained within the SK2-L protein. In mice that selectively lack the SK2-L isoform (SK2-Sonly) dendritic and spine expression is drastically reduced and the SK2 channel contribution to synaptically evoked EPSPs is abolished. These results form the basis for the overriding hypothesis of this application: The unique SK2-L N-terminal domain confers dendritic and spine localization and function to SK2 channels that is accomplished by selective association with targeting proteins. We will use an integrated repertoire of electrophysiology, immunoEM, molecular biology, and biochemistry to test the following specific hypotheses. 1. Hypothesis: The loss of either isoform alters CA1 cellular physiology and hippocampal-dependent learning. Use SK2-Sonly and SK2-Lonly mice to determine: A) the subcellular distributions of SK2-L and SK2-S in CA1 neurons;B) the effects of SK2-S and SK2-L on synaptic plasticity and EPSPs, and on SK2 internalization following the induction of LTP;C) memory encoding in hippocampal-dependent spatial and non-spatial learning tasks. WT, SK2-Sonly/SK2-Lonly, and SK2-/- mice will serve as controls. 2. Hypothesis: The SK2-L N-terminus contains determinants that are necessary and sufficient for dendritic targeting. A) Determine the subcellular distributions of SK2-S, or SK2-L without or with SK2-S expressed in hippocampal neurons from SK2-/- mice that lack both SK2 isoforms. B) i. Identify the domains responsible for dendritic localization;ii. Determine whether these motifs are autonomous. 3. Hypothesis: Proteomics using SK2-Lonly or SK2-Sonly mice will identify proteins that specifically co-assemble with SK2-L subunits and mediate dendritic targeting. Protein complexes associated with SK2 channels will be immunopurified from SK2-Lonly or SK2-Sonly mouse brain using an antibody specific for SK2-L, or a pan-SK2 antibody, respectively. Parallel experiments using SK2-/- mice will serve as a negative control. Proteins that specifically co-assemble with SK2-L will be identified by mass spectrometry and investigated for their functional significance. PUBLIC HEALTH RELEVANCE: SK2 channels, one type of Ca2+-activated K+ channel, influence learning and memory. Blocking SK2 channels facilitates learning in animal models of brain damage, and reverses the navigation failure in an animal model associated with the development of cognitive disorders in Alzheimer's disease as well as during normal brain aging. These studies will reveal novel mechanisms for how SK2 channels influence information processing and storage in the brain, and will suggest novel interventional strategies that target different isoforms of SK2 channels to treat learning deficits associated with trauma, pathology and normal aging.

DESCRIPTION (provided by applicant): Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) causes ischemia, neuronal excitotoxicity and cognitive decline. Despite intensive efforts, outcome remains poor. Excitotoxicity results from increased glutamate neurotransmission, and the consequent excessive Ca2+ influx through NMDA-type glutamate receptors (NMDAr). Hippocampal CA1 neurons are important to learning and memory and are acutely sensitive to excitotoxicity. We have shown that small conductance Ca2+-activated K+ channels, type 2 (SK2 channels) are expressed together with NMDAr in the spines on hippocampal CA1 neurons where they act to attenuate Ca2+ influx through NMDAr. In addition, SK2 channels are removed from synapses following patterned activity, either normally as for the induction of long term potentiation (LTP), or abnormally after CA/CPR. The loss of synaptic SK2 channels removes the SK channel 'brake'on Ca2+ influx through NMDAr and is due to protein kinase A phosphorylation of the SK2 channels. Our results further show that increasing SK2 channel activity substantially improves neuronal survival after CA/CPR. Therefore, we will use an integrated technical repertoire to test these specific hypotheses: 1. Genetic or pharmacologic enhancement of SK2 channel activity protects CA1 neurons and improves cognitive outcome. We will use genetic mouse models and SK enhancing drugs to determine the i) survival of CA1 neurons and, ii) cognitive performance. 2. CA/CPR-induced ischemia causes a delayed and prolonged loss of synaptic SK2 channels in CA1 neurons, increasing the NMDAr-dependent Ca2+ transient that causes excitotoxicity. Preserving synaptic SK2 channel activity after CA/CPR protects CA1 neurons. We will measure the time course and effects of ischemia on the SK2 and NMDAr contributions to glutamate transmission (EPSP), and NMDAr-mediated Ca2+ transients. 3. CA/CPR-induced ischemia causes PKA phosphorylation of spine SK2 channels, inducing channel endocytosis. Expression of PKA-immune SK2 channels will normalize the SK2 and NMDAr contributions to the EPSP, the NMDAr-dependent Ca2+ transient, and protect CA1 neurons from excitotoxic cell death. We will use control mice or mice expressing PKA-immune SK2 channels to determine: i) the sub-spine distribution of SK2 channels;ii) the SK2 and NMDAr contributions to the EPSP;iii) the spine Ca2+ transient;iv) CA1 viability. 4. The aberrantly sustained ischemia-induced loss of synaptic SK2 channels results in ischemic LTP (iLTP) that shifts ?m, the modification threshold, to higher stimulus frequencies and impairs further potentiation. Maintained expression of functional synaptic SK2 channels prevents iLTP and normalizes ?m. We will measure the long-term effects of CA/CPR-induced ischemia on synaptic plasticity. PUBLIC HEALTH RELEVANCE: Heart attack and the consequent cerebral ischemia is one of the leading causes of death and disability in the United States and, unfortunately, there are currently no drugs available that improve outcome following severe heart attack requiring cardio-pulmonary resuscitation. SK2 channels, one type of Ca2+- activated K+ channel, are anatomically and functionally poised to ameliorate brain damage following stroke. The proposed studies will demonstrate the neuroprotective role of SK2 channels and suggest novel interventional strategies to protect the brain following heart attack, improving survival, diminishing memory deficits, and improving quality of life.

DESCRIPTION (provided by applicant): The slow afterhyperpolarization (sAHP) that follows an action potential in many central and peripheral neurons is due to the activation of voltage-independent, Ca2+-activated K+ channels. Hippocampal CA1 neurons have served as models for studying the sAHP and the underlying current, the IsAHP. The results of studies performed over the past two decades show that the sAHP has a profound influence on neuronal intrinsic excitability, being responsible for spike-frequency adaptation that regulates burst frequency. The sAHP is one of the principal targets for the ascending modulatory neurotransmitter systems that are involved in regulating the sleep-wake cycle, arousal, attention, and in modulating sensory processing, behaviors, emotions and memory consolidation. Importantly, the (I)sAHP decreases following learning, increasing intrinsic excitability. In addition, the (I)sAHP increases with age, reducing intrinsic excitability, and this age-related increase plays an integral role in the learning impairments that accompany normal aging. A similar increase in the (I)sAHP occurs in Alzheimer's disease models. The (I)sAHP channels are defined by: Ca2+-dependence, voltage-independence, K+-selectivity, and invariant slow activation kinetics. Indistinguishable (I)sAHPs have been recorded from hippocampal CA1and CA3, layers II-III of the cortex, (lateral) amygdala, and (midline) thalamus. SK channels and M-channels have been suggested to form the (I)sAHP channels, but there is abundant contradictory evidence. Therefore, despite decades of work, the molecular identity of the (I)sAHP channels remains to be determined. We have used bioinformatic genome analysis coupled with the functional characteristics of cloned channels, results from knockout mice, and detailed cell-type expression data for all K+ channel genes to identify 2 high priority candidates for the (I)sAHP channels. We propose to use a combination of molecular biological and electrophysiological techniques to test these candidates and identify clones encoding the pore-forming subunits of the (I)sAHP channels. Determining the identities of the (I)sAHP channels will provide a powerful target for therapeutic approaches to multiple central pathologies such as Alzheimer's disease, schizophrenia, epilepsy, attention deficit syndrome, and sleep disorders, as well as for cognitive impairment during normal aging. PUBLIC HEALTH RELEVANCE: The slow afterhyperpolization (AHP) channels regulate intrinsic excitability in many central neurons, and their activity is important for normal sleep-wake cycle, arousal, attention, and in modulating sensory processing, behaviors, emotions and memory consolidation. We will clone the slow AHP channels and define their requisite components. Determining the identities of the slow AHP channels will provide a powerful target for therapeutic approaches to multiple central pathologies such as Alzheimer's disease, schizophrenia, epilepsy, attention deficit syndrome, and sleep disorders, as well as for cognitive impairment during normal aging.

DESCRIPTION (provided by applicant): Each year approximately 500,000 people suffer from cardiac arrest in the United States, an event associated with poor neurological outcome. Despite intense research over the past 50 years, there are no pharmacological interventions that have proven successful in improving survival and outcome. A hallmark of ischemia-induced neuronal death is excessive release of glutamate leading to excitotoxicity. Unfortunately, glutamate antagonists have proven unsuccessful in humans, predominantly due to side effects. The logical alternative approach would be to apply compounds that activate GABA-A receptors (GABA-A R) in order to counteract excessive glutamate release and excitotoxicity. Interestingly, GABAergic compounds have yielded disappointingly variable results. Recent data has demonstrated that ischemia results in a rapid loss of GABA-A R protein, indicating that the ischemia-induced decrease in GABA-A R protein may cause a decrease in efficacy of GABA-potentiating compounds. Therefore, a treatment that stabilizes GABA-A R protein and function during an ischemic event is an appealing and exciting new approach to neuroprotection. In order to obtain electrophysiological recordings of neuronal GABA-A R function following ischemia, we have developed a cerebellar neuronal culture model. We will use a combination of methods, most notably whole-cell voltage- clamp recordings of synaptic (mIPSCs) and total GABA-A R activity (Current in response to exogenously applied saturating GABA) to confirm and extend upon our preliminary observation that ischemia causes a reduction in functional GABA-A Rs and importantly that ALLO prevents this ischemia-induced loss of function. This RO1 application will test four specific hypotheses 1) that ALLO prevents ischemia-induced reduction in functional GABA-A R, thereby protecting PCs from ischemia. 2) ALLO prevents ischemia-induced reduction in GABA-A R function by maintaining PKC activity and phosphorylation of GABA-A Rs during ischemia. 3) ALLO stabilizes GABA-A R protein during ischemia by preventing proteosome-dependent degradation of GABA-A receptor protein following ischemia and finally 4) that the ALLO-induced protection of GABA-A R function occurs in intact animals exposed to global ischemia (cardiac arrest). Our findings will begin to elucidate the cellular mechanisms of ALLO neuroprotection of PCs and determine molecular pathways that may represent novel targets for neuroprotection.

DESCRIPTION (provided by applicant): Synaptic Ca2+-activated K+ channels, SK2 channels, influence neurotransmission, synaptic plasticity, and learning and memory. Blocking SK channel activity facilitates synaptic plasticity and learning and memory while overexpressing SK2 or pharmacologically increasing SK channel activity impairs these processes. We discovered the molecular and cellular mechanisms that are likely responsible for the effects of SK2 channels on synaptic plasticity, the leading model for cellular changes underlying learning and memory. We showed that the activity of SK2 channels in the dendritic spines of hippocampal CA1 pyramidal neurons is coupled to NMDAR activity. Synaptically evoked Ca2+ entry into spines activates synaptic SK2 channels that repolarize the spine membrane potential, thereby favoring Mg2+ re-block of NMDARs, and thus limiting Ca2+ influx through NMDARs that is crucial to the induction of synaptic plasticity. In addition we showed that plasticity-dependent trafficking of SK2 channels itself contributes to the expression of NMDAR-dependent long-term potentiation. New results suggest that SK2 channel trafficking is linked to NMDAR trafficking that is orchestrated and coordinated by a novel family of synaptic scaffolding proteins to affect synaptic dynamics. We will use an integrated repertoire of electrophysiology in fresh brain slice preparations and recordings from transfected cells, biochemical pull-down assays and reconstitutions experiments, and innovative immuno-electron microscopy to examine the molecular and cellular mechanisms that engender the orchestrated trafficking of SK2 channels and NMDARs. The results have profound implications for novel interventional strategies to treat a wide range of cognitive disorders. PUBLIC HEALTH RELEVANCE: Long-term synaptic plasticity is widely thought to be the cellular substrate for learning and memory. The proposed research will illuminate novel pathways and molecules employed by neurons in the brain to orchestrate cellular rearrangements that engender synaptic plasticity. Therefore, this work will reveal potential therapeutic targets for a wide range of cognitive and other brain disorders.

DESCRIPTION (provided by applicant): There is currently no way to correct disease-causing mutations in the nervous system without altering the physiological level of the endogenous mRNA. This is a serious challenge because haplo-insufficiency or two-fold over-expression is often sufficient to cause neurological disorders. An example is Rett Syndrome, caused by mutations in the Mecp2 gene. Mecp2 gene duplication, as well as loss-of-function, results in severe disease. We propose to meet the challenge by harnessing the natural ability of RNA editing enzymes to site-specifically fix mutations in endogenous mRNAs. As a target for gene therapy, mRNA offers advantages over DNA. Messenger RNA is cytoplasmic, a readily available substrate, and unlike DNA in which 'mistakes' will be maintained, mRNAs turnover, replenishing the therapeutic target. Our new approach, Site Directed RNA Editing (SDRE), offers enormous untapped potential for correcting mutations, particularly those affecting the nervous system, and for exploring fundamental biological questions. RNA editing, which occurs through adenosine or cytidine deamination, is a natural process. When it occurs within the coding sequence of an mRNA specific codons can be re-coded to produce an altered amino acid sequence. For example, excitatory neurotransmission absolutely depends on the editing of a single adenosine within AMPA-type glutamate receptor mRNAs. Recognizing the power of this activity, we engineered a hybrid modular adenosine deaminase. When used in combination with a small antisense guide RNA we can site-specifically target any chosen adenosine. A similar strategy will be employed to create a site-directed cytidine deaminase. Unlike established therapies that focus strictly on regulating gene expression, SDRE can also fine-tune protein function. Inherited mutations that underlie diseases due to amino acid substitutions or premature stop codons can be corrected, and second-site suppressor mutations that restore function can be selectively introduced. We will demonstrate the power of SDRE within the context of neurobiology, but importantly, it applies to any biological system.