Lawrence B. Salkoff - US grants

Potassium channels are a diverse group of ion channels which are involved in virtually all aspects of membrane electrical behavior. Potassium channels are virtually ubiquitous among the cells of eukaryotes and the most heterogeneous of the voltage-gated cation channels. Because K+ channels are universally involved in membrane excitability, it is likely that most aspects of behavior and higher brain function in animals involves potassium channels in some way. In order to understand the significance of the great molecular diversity of K+ channels to behavior and many aspects of cell biology, we need to know the full extent of this diversity. The first aim of this proposal is to identify the full "set" of genes encoding voltage-dependent potassium channels in Drosophila. The relatively tiny Drosophila genome presents an opportunity to define the complete set of potassium channels in a single animal. It is our hypothesis that voltage- gated ion channels evolved nearly optimal structures prior to the separation of vertebrate and invertebrate species. Thus, all higher forms of life may share the same essential "set" of excitable channels. To verify this we propose to clone and express from mouse, any new K+ channel genes first isolated in Drosophila. Additional aims are: 1. to reveal the biophysical properties of each type of cloned channel by using the Xenopus oocyte expression system; 2. to determine whether cloned K+ channel subunits define separate K+ channel subfamilies or whether they mix to form heteromultimeric channels with subunits from other genes; 3. to determine the in vivo properties of cloned K+ channels, and their cellular and subcellular distributions. Underlying the multiplicity of potassium channel genes is their heterogeneity of function; determining both the functional properties and cellular distribution of the different potassium channels may reveal insights into their function. The techniques to investigate these questions will involve genetics, Northern analysis, PCR and immunocytochemistry.

Potassium channels are a diverse group of ion channels which are involved in virtually all aspects of membrane electrical behavior. Potassium channels are virtually ubiquitous among the cells of eukaryotes and the most heterogeneous of the voltage-gated cation channels. Because K+ channels are universally involved in membrane excitability, it is likely that most aspects of behavior and higher brain function in animals involves potassium channels in some way. In order to understand the significance of the great molecular diversity of K+ channels to behavior and many aspects of cell biology, we need to know the full extent of this diversity. The first aim of this proposal is to identify the full "set" of genes encoding voltage-dependent potassium channels in Drosophila. The relatively tiny Drosophila genome presents an opportunity to define the complete set of potassium channels in a single animal. It is our hypothesis that voltage- gated ion channels evolved nearly optimal structures prior to the separation of vertebrate and invertebrate species. Thus, all higher forms of life may share the same essential "set" of excitable channels. To verify this we propose to clone and express from mouse, any new K+ channel genes first isolated in Drosophila. Additional aims are: 1. to reveal the biophysical properties of each type of cloned channel by using the Xenopus oocyte expression system; 2. to determine whether cloned K+ channel subunits define separate K+ channel subfamilies or whether they mix to form heteromultimeric channels with subunits from other genes; 3. to determine the in vivo properties of cloned K+ channels, and their cellular and subcellular distributions. Underlying the multiplicity of potassium channel genes is their heterogeneity of function; determining both the functional properties and cellular distribution of the different potassium channels may reveal insights into their function. The techniques to investigate these questions will involve genetics, Northern analysis, PCR and immunocytochemistry.

DESCRIPTION: This is a renewal application to continue studies on the molecular properties of potassium channels in Drosophila. By taking advantage of the relatively small genome size of the fruit fly, the investigator plans to define the entire complement of potassium channels in this organism, including voltage-dependent, calcium-dependent, and inward rectifier types. The guiding thesis in this proposal is that, in contrast to mammals, it seems that the fly has fewer members of each potassium channel subclass. One can therefore examine the specific contributions of each of the five known classes of potassium channels in the Drosophila system because there is no (known) complication due functional redundancy. In addition, the identification of a novel Drosophila potassium channel subclass may lead to the isolation of several novel mammalian homologues. The long-term plan is to obtain a 'blueprint' of the entire potassium channel system in the fly so that the contribution of any one channel subtype can eventually be related to its physiological and behavioral role in the adult organism. Novel potassium channel types that are first identified in flys will also be subsequently identified in mammals. Each of the cloned potassium channel subtypes will be functionally characterized in the Xenopus oocyte expression system and its cellular distribution within the fly will be determined by immunological methods. Finally, a reverse genetic approach using dominant-negative mutation strategy will be employed to identify roles of each channel subclass in fly development and behavior.

DESCRIPTION: This is a proposal to undertake a comprehensive study that will reveal the structure, expression patterns, and basic functional properties of all potassium channels in C. elegans. The project will provide the first glimpse of the entire K channel set in one animal, without favoring any cell, tissue type or abundance class, revealing aspects of genomic organization that are not discernible without the complete gene sequences. Initially, all sequence information revealed by the C. elegans genomic project at Washington University will be compiled, and then the expression patterns of individual K channels will be determined. Because there are an estimated 100 potassium channels in an organism with only 302 neurons, many of these channels may be under coordinated regulation for expression; this may be required for complex electrical behavior. This study may lead to a profile not only of structure an functional properties, but also tissue distribution and amount and timing of expression. These data may serve as a model for interpreting the human genome sequence data, and may also lead to clinical applications.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The C. elegans genome contains an extended gene family of potassium channels consisting of more than seventy members. Among these genes are an ancient conserved "set" of potassium channels with orthologues in both humans and Drosophila. Many of these conserved genes are of interest to researchers working in fields as diverse as cardiac physiology, neurological function, and kidney and digestive disorders. Thus, C. elegans has the potential of being a valuable model system for basic research in virtually all fields where excitable membranes and ion currents are important. The investigators propose to create a resource base that will include full-length potassium channel cDNA clones in vectors suitable for functional expression in mammalian cell lines and Xenopus oocytes, and stocks of gene KO strains. Supporting this resource base will be studies showing the basic biophysical properties of the channels, studies showing the phenotypes of KO mutants and information on the cell-type expression patterns of each channel. Many aspects of this comprehensive project will directly guide studies in mammals and should offer an alternative model system applicable to a wide variety of researchers in both basic and applied research. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A comparative genomic approach, which began with the analysis of the unusual slo-2 gene in C. elegans has led to the molecular identification of the long sought-after mammalian Na+-activated potassium channel (KNa). KNa channels have been identified in cardiomyocytes and neurons where they may serve as an important protective mechanism against ischemia (Dryer, 1994). Our studies showed that the C. elegans gene, slo-2, also confers resistance to hypoxia (Yuan et al, submitted). Our studies further showed that mammalian KNa channels are encoded by the rSlack gene (Joiner, et al, 1998) (rslo2), a mammalian orthologue of the C. elegans slo-2 gene. We showed that rSLO-2 channels have all the properties of native KNa channels. We now plan to follow up on these findings by investigating the role of SLO-2 channels in different classes of identified neurons (in C. elegans), cloning and functionally characterizing two distinct mammalian slo-2 genes present in human (and mouse) genomes, and defining the structural regions in mammalian SLO-2 channels which are responsible for the salient property of sensing Na + and CI-. In addition to extending our theoretical understanding of the factors involved in ion channel activation, this information may be important for clinical applications. Pharmacological agents which modulate the KNa channel, especially channel openers, might serve a useful role in preventing cell damage during cardiac ischemia and stroke, and may serve as a protective agent in the pretreatment of organs used in transplant procedures.

We believe that our most recent work studying the electrical properties of neurons of the brain represents a breakthrough in understanding how potassium channels control the excitability of neuronal electrical activity, and may have a significant impact on both basic and clinical neuroscience. We have recently shown that one of the largest components of delayed outward potassium conductance in many neuronal types, during normal physiology has gone unnoticed and is the product of Na+-activated SLO2 (Slack) channels. Previous studies of potassium conductances in mammalian neurons may have overlooked this large component of outward current because the Na+ channel blocker TTX is typically used in studies of mammalian K+ channels and TTX also removes SLO2 currrents as a secondary consequence of its block of Na+ entry. Since most prior studies of the electrical properties of CNS neurons have overlooked the large SLO2 component, we propose to show its contribution to the electrical properties of neurons in several brain regions where it is prominently expressed. This will allow a more thorough understanding of the currents that determine neuronal electrical activity and may reveal SLO2 channels as useful pharmacological targets for the control of epilepsy and other seizure disorders. Because SLO2 channels are prominently expressed in the striatum they may also be a useful target in the treatment of Parkinson's Disease and in the treatment of depressive illness. In addition to showing the contribution of SLO2 channels to neuronal electrical excitability, we will also reveal more about the mechanism of SLO2 K+ current activation. We previously discovered that the SLO2 K+ current is activated by Na+ entry through a persistent inward sodium conductance. Thus, we will determine the genetic identity of one or more sodium channels that carry such a persistent sodium current capable of activating SLO2 channels. We will also investigate the functional relationships between sodium channels which carry a persistent Na+ current, and SLO2 Na+-activated channels. These experiments will be undertaken in a heterologous system where we will reconstitute a SLO2-sodium channel coupled system, and in experiments using single membrane patches from native neurons where the functional interactions of sodium channels and SLO2 channels can be studied under circumscribed conditions where sodium entry is limited to the patch.

? DESCRIPTION (provided by applicant): The sodium-activated K+ current (IKNa) is present in many neurons of the mammalian brain. Using wave- clamp experiments we will test the hypothesis that IKNa is a significant but overlooked delayed outward current responsible for action potential repolarization throughout the brain. Our preliminary results suggest that IKNa is by far the largest K+ current in action potential repolarization in mitral cells. The importance of IKNa in controlling excitability and repolarizing action potentials in the nervous system has been overlooked because the sodium channel blocker TTX is often used to study K+ currents in neurons; we found that TTX blocks the component of INaP that activates IKNa. Thus TTX blocks IKNa as a secondary consequence of its blocking INaP. We recently showed that IKNa was functionally coupled to one or more persistent sodium currents, (INaP). Hence, another aim of this proposal is to attempt to reveal the molecular identity of the one or more channels producing INaP which is functionally coupled to IKNa. Although INaP is thought to be the product of voltage gated sodium channels (VGSCs), the exact identity of the individual VGSC alpha and beta subunits which produce INaP in particular neuron types, has not been well established. INaP, is the major target of most antiepileptic drugs. In neocortex, the prominent INaP in layer V pyramidal neurons, augments excitability in neurons that comprise the neocortical output circuits which are involved in the spread of epileptic activity. Thus, revealing the molecular identify of INaP in layer 5 pyramidal neurons might lead to drugs that more specifically and effectively target INaP leading to better control of epilepsy. We will also evaluae the role of IKNa in that cell type and others to reveal the extent to which the interaction of IKNa and INap is important in regulating excitability. These studies may also provide a better understanding of other neurological diseases where increased activity of INaP may be a factor such as Amyotrophic Lateral Sclerosis and Familial Hemiplegic Migraine; additionally, blockade of INaP may be a useful treatment for Multiple Sclerosis. Specific Aims are: 1. to attempt to determine the molecular identities of TTX-sensitive INaP in several neuronal types including layer 5 cortical pyramidal neurons; 2. to attempt to identify the INaP component(s) functionally linked to the sodium-activated potassium channel (IKNa); 3. to test the hypothesis that IKNa is a significant but overlooked factor in action potential repolarization in many neuronal types.

? DESCRIPTION (provided by applicant): This proposal tests the hypothesis that the unusual sodium-activated K+ current controls synaptic integration in an unanticipated way which is widespread in the mammalian brain. As such, it may be a previously unrecognized factor in memory and learning. Sodium-activated K+ channels (KNa channels) are present in most areas of the brain and are prevalent in the cell bodies of many pyramidal cells. In mitral cells of the olfactory bulb, KNa channels are densely packed into the plasma membrane of the cell body. In mitral cells, the cell body is interposed between the dendritic tuft which inputs synaptic potentials and the axon initial segment which is one site of action potential initiation in these cells. If KNa channels in the cell body were constitutively active, incoming synaptic potentials from the glomerulus would be shunted by the high K+ conductance of the soma, never reaching the axon spike initiation zone. However, we have shown by heterologous expression that KNa channels are highly modulated by metabotropic signaling through the G-alphaQ signaling pathway. If similarly modulated in mitral and other cells, their activity could be largely blocked r unblocked by metabotropic signaling at the soma. Thus, KNa channels may be gate-keepers of synaptic integration in the olfactory bulb where they could either block or permit the generation of action potentials by incoming synaptic potentials. This proposal aims to test the hypothesis that (1) KNa channels present in the cell bodies of mitral cells are indeed regulated by metabotropic signaling and (2) KNa channels must be down-regulated to permit the cell to generate an action potential. KNa channels are found in the plasma membrane of neuronal soma throughout the nervous system and could well be an important factor governing the intrinsic excitability of many cells; the intrinsic excitability of a neuron is increasingly undersood to be an important factor in synaptic integration and plasticity, and may be an important non-synaptic factor in memory and learning. These studies may provide a better understanding and treatment of disease which leads to deficits in learning and memory loss, such as the multiple syndromes producing senile dementia.

? DESCRIPTION (provided by applicant): High conductance Ca2+ and voltage activated K+ channels (Slo1 or BK channels) are widely distributed and play numerous physiological roles. BK channels function as ? subunits alone, as in skeletal muscle, or in association with auxiliary ? subunits (?1- ?4) where they confer diverse functional properties in different tissues. Defectve or missing BK channels or their ? subunits have been associated with many disease processes including hypertension, asthma, autism, mental retardation, obesity, and epilepsy. Understanding the normal mechanism of activation of BK channels is crucial to understanding how function is altered in disease, and to provide molecular information that would be useful in developing possible therapies. The four ? subunits of Slo1 assemble to form a channel with an intra-membrane Core and a cytoplasmic Tail. The Core consists of four voltage sensing domains (VSD) and a pore gate domain (PGD). The cytoplasmic Tails form a large intracellular gating ring. Ca2+ binding to the gating ring activates the PGD through a poorly understood coupling mechanism from gating ring to Core. Also poorly understood are the sites and mechanisms of action of the various ? subunits on BK channels. Two recent advances will allow us to apply new approaches to resolve the mechanisms of coupling and ? subunit action. The first advance is obtaining the protein crystal structures of the gating ring in the closed and open conformations, which suggests that Ca2+ binding to the gating ring induces a push to the Core under the VSDs resulting from elevation of the four alpha-B helices of the gating ring, and a simultaneous pull on the S6 segments in the PGD of the Core arising from movement of lever arms in the gating ring. The second advance was our isolation and functional expression of the isolated Core itself, which provides a tool to assign observed functions to Core, gating ring, or both. Based on these advances and functional data we hypothesize that a novel Push-Pull mechanism couples Ca2+-dependent activation from the gating ring to the Core. In Aim 1 we critically test the Push-Pull hypothesis for Ca2+-dependent coupling using mutations with expected outcomes based on the Push-Pull hypothesis. In Aim 2 we use these advances to localize the sites of action of ?1- ?4 subunits on modifying gating of BK channels to the Core, gating ring, or both. In Aim 3 we seek to obtain the protein crystal structures of the mutated gating rings that alter Ca2+-dependent coupling and also the structures of the sites of contact between peptides of ? subunits and gating ring to provide structural insight into mechanism. The completion of these aims should provide new insight into the mechanism of Ca2+-dependent coupling between gating ring and Core, and also into the mechanisms for ? subunit modulation of BK channels. The Push-Pull model, if found to be consistent with the critical tests to be applied, will necessitate a paradigm shift in the proposed mechanism of Ca2+-dependent coupling, from a single active coupling structure to dual simultaneously active Push-Pull coupling structures.