Leonard K. Kaczmarek - US grants

Changes in the amplitude and kinetics of ionic currents within nerve cells of both vertebrate and invertebrate origin may be brought about by the activation of endogenous protein kinases. Transformation of the electrical properties of neurons by these enzymes may lead to both natural and pathological changes in excitability, such as the onset of neuronal afterdischarge. The research proposed in this application will use a simple invertebrate system of neurons, the bag cell neurons of Aplysia, which are accessible for combined biochemical-electrophysiological investigations to study the role of cyclic nucleotide, calcium/calmodulin and calcium/phospholipid dependent protein kinases in the control of calcium and potassium currents. Recent work has indicated that the amplitude of the calcium current in the bag cell neurons may be regulated by the activation of the calcium/phospholipid dependent protein kinase (protein kinase C). This hypothesis will be tested by voltage clamp studies in isolated, internally dialyzed neurons after injection of protein kinase C and, using single channel recordings, before and after microinjection or pharmacological activation of this enzyme. The electrical effects of the second messenger, inositol trisphosphate, which is believed to be formed concurrently with the activation of protein kinase C, will also be investigated. Parallel biochemical experiments will be carried out to determine the conditions under which activation of these enzymes occurs and the phosphoprotein substrates whose phosphorylation state is altered on the activation of these enzymes in intact cells. In addition, the mechanism by which cyclic AMP dependent protein kinase acts on three distinct potassium currents in the bag cell neurons will be investigated further using whole cell and single channel recordings.

Voltage-dependent potassium channels play a central role in setting the pattern of spontaneous firing and the shape of action potentials of muscle cells and neurons. Alterations in the level of expression and in the electrical characteristics of potassium channels occur in response to hormones, as well as to drugs that influence second messenger pathways linked to protein kinases. This project will investigate the mechanisms that regulated two different potassium channels whose genes have been isolated and have been termed the minK gene and the Kvl gene. Both genes are expressed in heart cells as well as in other tissues. MinK RNA, when injected into Xenopus oocytes, generates slowly activating potassium currents whose amplitude is greatly enhanced by activation of the cyclic AMP-dependent protein kinase. Electrical recordings and biochemical measurements on normal and mutant minK channels expressed in heterologous cells will then be carried out to determine whether the channel is phosphorylated directly and whether modulation of channel activity occurs through the rapid recruitment of new channels to the plasma membrane. Mammalian cells lines that normally express minK RNA and protein will be identified and the physiological role and regulation of the channel will be investigated in such cells. A second series of experiments will be carried out with Kvl, a mammalian member of the Shaker family of proteins. Although a number of channel genes belonging to this family have been identified, no gene has yet been shown to account for a specific component of potassium current in a normal cell. Hybrid arrest techniques will therefore be used to determine whether the Kvl protein contributes to the voltage-dependent potassium current in GH3 cells, a clonal pituitary cell line that expresses Kvl mRNA at high levels. Electrophysiological measurements, coupled with biochemical studies of the Kvl protein identified with Kvl-specific antisera, will then be used to study the short-term and long-term regulation of the Kvl channel by hormones and second messengers.

ABSTRACT Spinocerebellar Ataxia Type 13 (SCA13) is caused by mutations in KCNC3, the gene that encodes Kv3.1 voltage-dependent potassium channels. This condition results in motor abnormalities and the inability to locate sounds in space. Kv3.3 is highly expressed in the cerebellum and in auditory brainstem nuclei, including the calyx of Held presynaptic terminals in the medial nucleus of the trapezoid body (MNTB). Kv3.3 differs from other closely-related channels in having an extended C-terminal cytoplasmic domain that recruits several cytoplasmic signaling molecules, including Tank Binding Kinase 1 (TBK1). This enzyme keeps Kv3.3 bound to Hax-1, a cell survival protein that, when bound to the channel, triggers the formation of a dense actin cytoskeleton under the plasma membrane. This process is impaired in a Kv3.3 mutant (G592R Kv3.3) that causes late-onset SCA13. This mutation overstimulates TBK1 activity but prevents the channel from triggering actin nucleation. The experiments in this proposal will test the hypothesis that TBK1 is required for normal synaptic transmission and endocytosis of synaptic vesicles because it is a physiological regulator of the interactions between the Kv3.3 and the underlying actin cytoskeleton. Patch clamp studies will be combined with imaging and EM immunomicroscopy to test the effects of TBK1 inhibition or manipulation of TBK1 activity with genetic approaches in cell lines and in the calyx of Held terminals from wild type, Kv3.3-/- and G592R Kv3.3 animals . Phospho-specific antibodies will be used to determine if TBK1 activity is altered by stimulation of auditory neurons in brain slice preparations and by acoustic stimulation of intact animals in vivo. Finally, the specific domains required to couple TBK1 to the Kv3.3/Hax-1 complex will be defined by mutagenesis studies and by proteomic approaches that identify phosphorylation sites for TBK1 in the channel complex. Our findings will provide novel insights into the regulation of synaptic transmission in normal and pathological conditions and will generate new targets for the treatment of diseases such as Spinocerebellar Ataxia Type 13 and other conditions that affect central auditory processing.

This proposal requests funds towards a complete renovation and approximately 11,224 square feet of research space in the Sterling Hall of Medicine of Yale University. The space represents just under half of that assigned to the Department of Pharmacology. There has been no renovation if many of these laboratories since 1965, and as a result much of it is in poor condition. Research in the department of pharmacology falls into three main ares, neuropharmacology, signal transduction and cancer pharmacology. The redesign of the proposed space would bring together the investigators in neuropharmacology, who have a strong focus on the molecular, biophysical and regulatory properties of ion channels and transporters. The faculty in this area are Barbara Ehrlich (biophysical properties of calcium release channels on intracellular membranes and their regulation by second messengers), Ed Moczydloski (biophysics of calcium-activated potassium channels and voltage-dependent sodium channels), Gary Rudnick (function of neurotransmitter transporters), Priscilla Dannies (regulation of peptide secretion in pituitary cells), Murdoch Ritchie (role of ion channels in glial cells) and Leonard Kaczmarek (regulation of prolonged changes in neuronal excitability by protein kinases and changes in gene expression). The proposed plan would allow these investigators to share open-plan laboratories, and would therefore encourage direct exchange of information, formal collaborations, and joint-mentoring of post-docs and students.

DESCRIPTION (provided by applicant): A class of calcium-activated potassium channels termed BK(Ca) channels are enriched at presynaptic neurotransmitter release sites within the brain, and are believed to control both the amount and timing of neurotransmitter release. BK(Ca) channels are encoded by the Slo gene. Recent evidence suggests that a related gene, Slack, also contributes to BK(Ca)-like channels in neurons, and that some BK(Ca) channels may be comprised of heteromultimers of Slo and Slack channels subunits. The experiments in this proposal will characterize the molecular, biophysical and structural properties of BK(Ca) channels in both the somata and presynaptic terminals of native neurons, and in transfected cells. The role of the Slo and Slack channel subunits in normal synaptic transmission, and the mechanisms of their response to hypoxia, will be determined by patch clamp measurements coupled with genetic knockout approaches. The structure and function of the large carboxy-terminal regulatory region of the Slo channel subunit with its calcium-binding sites, and its interaction with other proteins, including the Slack subunit, will be determined by biophysical and biochemical measurements and by X-ray crystallographic methods. Finally the structure of intact BK(Ca) channels will be determined by cryo-electronmicroscopy. Knowledge of the molecular properties and regulation of BK(Ca) channels in normal and hypoxic neurons, together with structural determinations of their interactions with both drugs and natural ligands, will be key in the development of more effective treatments for disorders such as epilepsy and stroke.

DESCRIPTION (provided by applicant): Many animal behaviors, including feeding and reproduction, are controlled by neurons that release neuropeptides. The occurrence and frequency of such behaviors is determined by the firing pattern of such neurons. This project will investigate the mechanisms that regulate the excitability of the bag cell neurons of Aplysia, a group of neurons that controls reproductive behaviors lasting several hours. In response to brief synaptic stimulation, these neurons generate about 30 minute after-discharge that triggers the release of neuropeptides. This is followed by a prolonged period of inhibition, termed the refractory state, which persists for about 18 hrs and limits the occurrence of the behaviors. We plan to determine the cellular mechanisms that produce the refractory state, which is associated with a prolonged enhancement of potassium currents. The contribution of two splice variants of the SIo gene, which encode calcium-activated potassium channels, to the refractory state will be determined using native neurons, heterologous expression and RNA interference approaches. Immunochemical experiments will establish whether the SIo channels are coupled to protein kinase C-regulated calcium channel subunits, which are localized to neuropeptide release site and may be modulated by insertion and removal from the plasma membrane. Secondly, we plan to test the hypothesis that recruitment of a Kv2-family potassium channel to ring-shaped clusters on the plasma membrane also contributes to the onset of refractoriness. Finally, we plan to investigate a protein kinase C-regulated, non-selective cation channel that provides the depolarization for afterdischarge. By molecular cloning and genetic manipulations, we plan to test the hypothesis that this channel is a member of the pkd and/or trp family of cation channels. We shall also determine whether the physical association of the channel with protein kinase C becomes altered during the refractory state. An understanding of how ion channels are regulated in peptidergic neurons may lead to an understanding of normal and pathological changes in behavior, and provide insights into pathological changes of excitability such as epileptic seizures.

DESCRIPTION (provided by applicant): The Slack and Slick genes encode Na+-activated K+ channels, which regulate the rate at which neurons adapt to maintained synaptic stimulation and the accuracy of timing of neuronal action potentials. These channels have also been proposed to play a key role in the protection of neurons and cardiomyocytes from hypoxic injury. In their general structure, they resemble other voltage-gated K channels, but have very large (>600 amino acid) intracellular C-termini. The C-terminal domain of Slack interacts with Fragile- X Mental Retardation protein (FMRP), an RNA-binding protein that regulates trafficking and translation of a subset of subset of neuronal mRNAs. The work in this application will use biochemical and electrophysiological assays to determine the specific regions of Slack and FMRP involved in their interactions both in vitro and in vivo, and will determine which specific regions are required for FMRP to control the gating of Slack channels. We shall also determine how the expression of Na+-activated K+ channels is altered in a mouse model of Fragile X syndrome (Fmr1-/-). In particular we shall determine the mechanism that in Fmr1-/- animals causes the total loss of expression of the distal C- terminus of Slack, a region that appears to be required for FMRP binding to the channels. Parallel electrophysiological and pharmacological experiments will evaluate the effects of this altered pattern of Slack expression on the functional properties of Na+- activated K+ channels in native neurons. An understanding of the biological properties and regulation of Slack and Slick channels will lead to a clearer understanding of the deficits in Fragile X syndrome and related disorders and is expected to lead to the development of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Recent evidence suggests that activation of a relatively newly discovered class of proteins, termed sodium- activated potassium channels is regulated by their binding to Fragile X Mental Retardation Protein (FMRP). Inherited loss or deficits in FMRP are the leading cause of inherited intellectual disorders. The experiments in this application will determine the mechanisms and biological consequences of this channel-FMRP interaction. This information will be used to evaluate whether these channels are likely to be therapeutically useful drug targets, and in particular, whether pharmacological manipulations of sodium-activated potassium channels can overcome some of the impairments in neuronal activity that accompany loss of FMRP.

DESCRIPTION (provided by applicant): Sodium-activated potassium (KNa) channels are widely expressed throughout the central nervous system. Activation of these channels is known to protect cells from hypoxic injury. The molecular correlate of KNa currents, however, was unknown until the genes underlying this new family of K+ channels were cloned relatively recently. Slack (Sequence like a calcium-activated K channel) and Slick, which are also referred to as Slo2.2 (KCa4.1) and Slo2.1 (KCa4.2), currently have no pharmacological tools that allow for modulation of their function. With the help of this grant we are therefore proposing to design potent and brain-penetrant Slack channel activators that could be used to explore the therapeutic potential of these interesting channels. In normal neurons, KNa channels contribute to the slow afterhyperpolarizations that follows repetitive firing, regulate rates of bursting and enhance the accuracy with which action potentials lock to incoming stimuli. Evidence further indicates that KNa channels play a crucial role in protecting cells from injury under ischemic conditions, when inhibition of the plasma membrane Na+-K+-ATPase by the lack of oxygen leads to an increase in intracellular sodium levels. Activation of KNa channels under these circumstances is likely to prevent calcium entry by stabilizing the membrane potential and protecting neurons from overloading with calcium. In proof of this concept, mutation of the ortholog of Slack in the nematode C. elegans renders these animals hypersensitive to hypoxia indicating that KNa channels provide endogenous protection against hypoxia in this species. Compounds that increase the activity of KNa channel therefore should be therapeutically useful for the treatment of stroke and the prevention of the effects of global cerebral ischemia as occurs, for example, in cerebral palsy. By increasing the slow afterhyperpolarizations, KNa channel activators may also be useful for reducing neuronal excitability in epilepsy and ataxia. By screening various pharmacophores known to activate the related large-conductance Ca2+-activated K+ channel BK (Slo1, Maxi-K) it was recently discovered that biphenylthioles and 4-arylquinolinones activate Slack channels in the low micromolar range. Interestingly, two compounds in the 4-arylquinolone series were found to increase Slack activity without exerting effects on BK channels demonstrating that it is possible to separate the two activities. By combining i) classical medicinal chemistry, ii) a recently developed high- throughput assay measuring mass redistribution at the plasma membrane to determine Slack activation, iii) electrophysiology and iv) pharmacokinetic experiments in rats we here propose to improve the potency, selectivity and brain-penetration of our leads. Our overall goal is to provide the scientific community with a Slack channel activator that is suitable for in vivo use. PUBLIC HEALTH RELEVANCE: Based on their abundant expression in the brain sodium-activated potassium (KNa) channels potentially constitute novel drug targets for the treatment of stroke, cerebral palsy, epilepsy and ataxia. However, these important channels currently have no pharmacological modulators. With the help of this grant we will attempt to design small molecule KNa channel activators that could be used as scientific tool compounds to test whether KNa channels indeed constitute novel targets for neurological diseases.

DESCRIPTION (provided by applicant): Auditory brainstem neurons fire at very high rates with extraordinarily high temporal precision, allowing them to encode specific features of sound stimuli. Unexpectedly, it has been found that the levels and characteristics of potassium channels in these neurons are rapidly modified by the auditory environment. We plan to determine the mechanisms of this use-dependent plasticity for two classes of K+ channels, i) voltage-dependent Kv3.1b channels and ii) Na+-activated K+ channels (KNa channels) encoded by the Slack and Slick genes. The function of Kv3.1b channels is to allow neurons to fire at high frequencies. We plan to determine whether sound- induced increases in Kv3.1b protein levels result in higher Kv3.1b currents in the plasma membrane and enhance the ability of MNTB neurons to fire at higher rates. We also plan to test the hypothesis that KNa current amplitude is regulated by sound-induced changes in phosphorylation state to enhance the temporal accuracy of these neurons at high rates of stimulation. For both Kv3.1b and KNa channels we will determine whether the activity-dependent increases in current are regulated by the Fragile X Mental Retardation Protein (FMRP), a repressor of protein translation that binds Kv3.1 mRNA and that modulates KNa channels by direct protein- protein interactions. An understanding of how the excitability of auditory neurons is regulated by physiological stimuli is likely to lead to novel pharmacological treatments for disorders of auditory function including tinnitus, age-related hearing loss and audiogenic seizures, as well as Fragile X syndrome and other disorders of excitability. PUBLIC HEALTH RELEVANCE: Recent evidence has shown that the excitability of auditory neurons within the brain is rapidly modified by changes in the ambient acoustic environment. The experiments in this proposal will determine the biochemical and biological mechanisms that adjust the properties of ion channels in these neurons, allowing them to maintain high accuracy in noisy environments. This information will be used to determine which classes of pharmacological agents can be used for the treatment of disorders of auditory function including tinnitus, age- related hearing loss and audiogenic seizures, as well as Fragile X syndrome.

Human mutations in KCNT1, the gene for the Slack Na+-activated K+ channel result in several different childhood epilepsies, including Malignant Migrating Partial Seizures in Infancy (MMPSI) and Autosomal Dominant Frontal Lobe Epilepsy (ADNLFE). These mutations are gain-of-function mutations that result in enhanced channel opening. Although the seizures may abate with adulthood, all disease-causing Slack mutations are associated with very severe intellectual disability. The intellectual deficits may result from the fact that the large intracellular C-terminus of Slack channels interacts with several cytoplasmic signaling molecules, including Phartr-1, Fragile-X Mental Retardation protein (FMRP) and Cytoplasmic FMRP-Interacting Protein (CYFIP1). The two latter proteins are well known regulators of mRNA translation in neurons. We will record from cortical neurons in cultures and in brain slices from mice expressing the human mutation R455H Slack, to determine how the firing patterns of neurons are altered to produce increased excitability, interictal spikes and spontaneous seizures. We will test the actions of a novel inhibitor of Slack channels to determine whether it reverses the effects of the mutation on neuronal firing and seizures and as well as altered patterns of behavior in the R455H mutant animals. Finally we will determine whether the interactions of Slack channels with their cytoplasmic signaling partners are disrupted in the mutant animals, and whether the ability of activation of Slack channels to modulate mRNA translation in neurons is compromised. This work will provide a biological basis for treatment of these devastating diseases and provide potential lead compounds for therapeutic intervention.

Cerebellar ataxia is a progressive neurodegenerative disease with major impact on the daily life of affected people, their families, and, on the health care system. There is no cure for cerebellar ataxia regardless of its origin. There is no effective treatment to slow the progression of symptoms that frequently lead to premature death. Ion channels have been implicated in the etiology of several form of cerebellar ataxia. For example, specific mutations in KCNC3, the gene encoding the Kv3.3 channel, such as the G592R point mutation, have been identified as the underlying genetic cause of late-onset spinocerebellar ataxia. Despite of these developments, there is no understanding of why late-onset cerebellar ataxias emerge in some but not all subjects with genetic mutations. There is also great variability in when during the lifespan of the affected subjects the symptoms emerge. Based on our unexpected preliminary observations, we hypothesize that disease onset in genetically vulnerable subjects is determined by host-pathogen interactions with particular emphasis on viral infections. Specifically, we assert that the synergistic activation of an intracellular signaling pathway that is targeted by both the mutant potassium channel and by viral defense mechanisms, impairs cellular function and results in the death of Purkinje cells. Our hypothesis and preliminary data challenge contemporary views on the etiology of cerebellar ataxia with implications for novel therapeutic strategies. We will test our hypothesis using cellular- and animal models with state-of-the-art neurobiological, molecular biological and immunobiological approaches.