David E. Clapham - US grants

Action potentials regulate the function of the heart by determining beating rate and contractility. The action potential in turn is generated by a population of ionic channels which undergo conformational changes to alter the permeability of the membrane. Much of our present understanding of the ionic currents which regulate heart function has been acquired through measurement of macroscopic currents under voltage clamp. Researchers have recently developed a technique measuring the current from small patches of membrane containing one to a few ionic channels. The purpose of the present research is to measure single channel currents from seven-day chick embryonic heart cell aggregates and single cells. The patch clamp technique allows measurements of single channels to be made from intact and excised patches of heart cell membrane. Single cells can also be voltage clamped to compare with conventional two-electrode voltage clamp. Attention will be focused on outward currents in the plateau and pacemaker ranges of membrane potential. Specific ionic channels will be identified by analysis of (1) single channel reversal potentials, (2) time constants, (3) conductance, and (4) pharmacology. Standard two-electrode voltage clamp and single cell voltage clamp recordings in the pacemaker and plateau range will be used to define total membrane currents. The effect of ionic concentrations and channel-blocking agents will be studied in excised membrane patches. The extracellular and intracellular face of the heart cell membrane may be selectively exposed. The selectivity of putative potassium channels to varying ionic concentrations will be measured. Potassium channel blocking agents will be compared for effects on channel conductance and kinetics. By combining techniques, a more detailed understanding of cardiac membrance currents regulating the pacemaker and plateau potentials may be gained. The proposal requests support for three years.

GTP-binding (G proteins) proteins are a key link between many cell surface receptors and the cellular response. They act as signal transducers in functions as diverse as control of heart rate and contractility, vision, cell division, control of exocytosis, ribosomal protein synthesis, and olfaction. There are three components of signal transduction in these systems: (1) receptor binding by a specific agonist which catalyzes the (2) splitting of the G protein heterotrimer into alpha-GTP and beta gamma subunits followed by (3) action of the subunits on specific effectors. Examples of effectors are the muscarinic-gated inwardly rectifying potassium (K+) channel in heart, adenylyl cyclase, and phospholipase C. Because there are several types of G protein alpha subunits and fewer (and less well understood) beta gamma subunits, the alpha subunits have been suspected to be the main activation arm of the G protein system. The cardiac muscarinic K+ channel will serve as the assay system to study receptor-G protein and G protein-channel interactions. The following questions will be addressed: (1) What are the G protein subunits that cause the cardiac K+ channel (iK.ACh) to open? We have strong evidence that beta gamma subunits open iK.ACh but we would like to explore the specificity of this action using mammalian heart G proteins on a mammalian atrial membrane. Other alpha and beta gamma subunits from various preparations will be used (including alpha41, alpha40, alpha39) and several tests for specificity of action performed. (2) What are the functional sites of both alpha and beta gamma subunits that allow receptor and/or channel interaction? Chemical modification and antibody binding will be used to modify structure. The functional consequences of these modifications will be compared to controls. (3) Is receptor specificity a function of receptor-G protein interaction? Using a Chinese hamster ovary cell line, four subtypes of muscarinic receptors will be expressed. The link between receptors and ion channels will then be explored using a combination of patch clamp and biochemical techniques. Steps will include characterization of ion currents in CHO cells, search for activation of channels by G protein subunits, and reconstitution of the heterotrimer between receptor and channel. (4) What is the mechanism of desensitization of the K+ current to muscarinic activation? In most cases, inside-out patches of cell membranes will be exposed to intracellular ligands, such as G protein subunits, to determine the steps from receptor binding to channel activation.

This proposal requests support to study the intracellular regulation of potassium-selective (K+) ion channels in heart cells. Ion channels are intramembrane proteins giving rise to the cardiac action potential and thus govern the rate and force of contraction of the heart. Cardiac arrhythmia, a leading cause of cardiovascular deaths, are caused by disturbances in this electrical control. Even more importantly, ion channels control the beat-to-beat regulation of the heart and allow the heart to respond to hormones and neurotransmitters and meet ever changing biological demands. The present proposal focuses on K+ channels they are highly regulated by intracellular second messengers. The large diversity of K+ channels reflects their importance in controlling many aspects of cardiac function. Work over the past several years has dramatically expanded our knowledge of the number of K+ channels and their intracellular regulation. Much of this work has been accomplished with patch clamp techniques in combination with reconstitution of biomedical pathways in the patch or intracellular environment. However, many of the details of ion channel regulation cannot be elucidated with these techniques alone. This is especially true for multistep, membrane-bound second messenger systems where specific inhibitors of the steps are lacking. This application proposes a continuation of electrophysiological and biochemical reconstitution approaches to further elucidate the control of several important K+ channels, including the muscarinic-gated inward rectifier, K.ACh, and two newly discovered K+ channels (IK.AA, IK.PC) gated by lipophilic membrane constituents. In particular, the lipoxygenase pathway's participation in control of IK.ACh will be explored and its physiologic relevance tested. The participation of phospholipid metabolic pathways in the control of IK.AA and IK.PC will be examined. In addition, molecular biology techniques will be employed to determined the primary structure of cardiac potassium-selective ion channels. Three cloning strategies to sequence voltage-sensitive and relatively voltage-insensitive K+ channels will be applied and the channels expressed in Xenopus oocytes and mammalian cell lines. The ability to alter specific regulatory sites in the primary structure of K+ channels will help provide the needed specificity to dissect the regulatory processes.

An international symposium on Organellar Ion Channels and Transporters will be held in September 1995 at the Marine Biological Laboratory in Woods Hole, Massachusetts, under the auspices of the Society of General Physiologists. The symposium will focus on the biochemistry, molecular biology, structure/function, and physiology of ion channels and transporters found in intracellular membranes such as endoplasmic reticulum, nucleus, and other organelles. The overall aim of the symposium is to integrate the physiological, biochemical, and genetic approaches to the study of this class membrane proteins and provide a focus for discussion of this exciting new frontier of membrane biology. In addition to the keynote address, there will be 24 lectures distributed in five sessions with the following themes: l) Endoplasmic reticulum ion channels and transporters; 2) Mitochondrial channels and transporters; 3) Nuclear channels and transporters; 4) Secretory mechanisms; and 5) Organellar localization of calcium. The speakers will organize their talks to address a mixed audience whose interests will range from protein chemistry to cell physiology. Twenty-four speakers, including-five from abroad, have formally agreed to participate. In addition to the lectures, we will have two poster sessions for contributed papers and one session (New Ideas, New Faces) of shorter talks by young investigators selected from the abstracts submitted for the poster session. Two keynote addresses will be delivered; the first by Dr. Clara Franzini-Armstrong of the University of Pennsylvania who will discuss ryanodine receptor morphology and the second by Dr. Erwin Neher of the Max-Planck-Institute of Gottingen, Germany, who will discuss regulation of intracellular calcium and control of secretion. We have made a special effort to include women speakers; seven of the speakers, including one keynote speaker, are women. The proceedings will be published by the Rockefeller University and has a committed distribution of over 1800 volumes. We expect this publication to constitute a compendium of novel approaches which will influence future research in this field.

The heart beats at an intrinsic rate which is modulated by sympathetic and parasympathetic innervation. The parasympathetic innervation, via the vagus nerve, slows heart rate and modulates contractility by secreting acetylcholine onto muscarinic reports (largely m2 subtype) on pacemaker cells of the heart. The bound muscarinic receptor activates a pertussis toxin-sensitive G protein heterotrimer (G alpha beta gamma) which, in turn, activates an inwardly rectifying K+-selective channel. The steps in G protein subunit(s) activation of the channel, I/KACh, have been the subject of intense investigation, not only because the channel is an important therapeutic target for control of supraventricular arrhythmias, but also because it is a unique model system for understanding G protein- mediated signal transduction. We propose to define the molecular mechanisms underlying G protein regulation of the muscarinicgrated, inwardly-rectifying potassium channel, I/KACh. Recently, two rat atrial cDNAs (GIRK1 and KGA) have been cloned, and when expressed, exhibit many of the properties of I/KACh. Also, only recently have functional recombinant G protein beta-gamma subunits (G beta gamma) been purified and characterized. These two breakthroughs will enable us to systematically investigate the molecular structures regulating I/KACh. The experiments we propose are based on work from our laboratory which indicates that G beta gamma is the physiological mediator of I/KACh activation. There are three specific aims. First we will characterize heterologously- expressed GIRK1 in Xenopus oocytes, Sf9 cells, and GIRK1-transfected Chinese hamster ovary (CHO) cells to lay the groundwork for channel mutagenesis. Our second aim is to determine the molecular structures in GIRK1 interacting directly with G beta gamma. We hypothesize a direct interaction between G beta gamma and GIRK1, and based on a comparison of GIRK1 with the G protein-insensitive IRK1, we speculate that the carboxyl-terminal portion of GIRK1 is the G protein regulatory region. We will make chimeras of IRK1 and GIRK1, and express GIRK1 deletion mutants to test this hypothesis. We will express and purify whole channel and cytoplasmic channel fragments from baculoviral and E coli expression systems and use electrophysiological and biochemical approaches to localize the region of GIRK1 that interacts with G beta gamma. Third, we will explore potential effector interaction domains within G beta gamma. The structure-dependent function of G beta gamma is unknown since G beta gamma effectors have only recently been identified. We will modify residues in G gamma to alter its isoprenylation and compare these altered G beta gamma s for their ability to activate I/KACh and GIRK1. Finally, will alter the G beta subunit to test for potential channel binding domains in the G beta region of the molecule.

Intracellular calcium (Ca2+) regulates or controls cell functions as diverse as proliferation, secretion, contraction, motility, metabolism, and gene expression. In the last ten years, scientists have discovered that many cell surface receptors initiate a cascade of events which result in the release of Ca2+ from intracellular stores, such as the endoplasmic reticulum. The unexpected dynamic behaviours of regenerative Ca2+ release, buffering, reuptake, and gating of Ca2+ entry from the extracellular space are only beginning to be studied and are still poorly understood. We propose to focus our research efforts on four related aspects of Ca2+ regulation. The four aims of the proposal are to l) define the initial steps in the receptor-dependent release of Ca2+ by specific G proteins; 2) measure the distribution and function of one Ca2+ release channel, the inositol (1,4,5) triphosphate (IP3) receptor, on intracellular stores. We have made the first direct recordings of this channel on intracellular membranes and will study its regulation in vivo. Exciting preliminary findings on the regulation of nuclear calcium will be extended; 3) better define the identities and functions of intracellular Ca2+ buffers; and 4) identify, clone, and examine the regulation of Ca2+ entry channel pathways that replete intracellular Ca2+ stores. The Xenopus laevis oocyte will be the focus of our studies. The oocyte has many advantages for study of Ca2+ regulation including ease of experimental manipulation and simplicity. Cloned keys proteins in Ca2+ regulation are highly homologous to mammalian cells. Finally, substantial evidence has accumulated that Ca2+ regulation in these cells closely follows that of many inexcitable mammalian cells.

technology /technique development; animal tissue; mental disorders; cognition; microscopy; lasers; nervous system; human tissue; biomedical resource; biomedical equipment development;

Dendritic spines are the main receipts of excitatory synapses in the central nervous system. One of the main hypothesis trying to explain the physiological role of dendritic spines claims that these small dendritic structures form a biochemical compartment for intracellular calcium. According to this hypothesis activation of synaptic inputs innervating a spine induced a localized increase of the inti-acellular calcium concentration in the spine mediated by influx of calcium via NMDA receptor channels or metabotropic glutamate receptor mediated release of calcium from intracellular stores. The synaptic induced spine calcium transients are thought to mediate long term changes in synaptic efficacy such as long term potentiation and depression, which may play a key role in m-emory and learning. Previously no intracellular biological active substance was uncaged by a two-photon laser in brain slices. Presently we are in the final stages of setting up a system which will allow simultaneous two photon uncaging and imaging in brain slices. With the use of this set up we plan to perform several experiments addressing the role of dendritic spines as calcium compartments, and their possible role in long term synaptic plasticity. 1) Uncaging of calcium in dendritic spines and neighboring parent dendrites. In this project we will characterize the diffusion of calcium into and from the spines, and thus examine the role of dendritic spines as localized calcium compartments. Recently the diffusion of uncaged fluorescein was examined in dendritic spines, but the relevance of these experiments to intracellular calcium in dendritic spines is unclear. 2) Uncaging of IP3 in dendritic spines and neighboring parent dendrites and characterization of the calcium transients resulting from IP3 mediated release of calcium from intracellular stores.

The vagus nerve slow heart rate in part by activating a cardiac potassium (K+) channel, I/KACH- Activated muscarinic receptors release the G protein Gbetagamma dimer which dramatically increases channel activity by up to 1,000-fold. We showed that I/KACH was composed of two distinct inwardly rectifying K+ channel subunits, KIRK1 (Kir3.1) and GIRK4 (KIR 3.4). In this proposal we outline the experiments that over the next five years will reveal the assembly expression, and stabilization of the cardiac I/KACh channel. First, we will use a combination of electrophysiology, biochemistry, and molecular biology to study how the GIRK1/GIRK4 subunits assembly and determine proteins associating with the final complex. In particular we are interested in how a particular receptor maintains its specificity of activation of the channel. Second, evidence indicates that key lipids are important to the regulation and stability of the final channel complex once it has reached the plasma membrane. We will refine our understanding of the role of Gbetagamma and phosphatidylinositols in the regulation of the functional GIRK1/GIRK4 complex. Finally, we will characterize the promoters of GIRK channel subunits in preparation for defining transcriptional control of their expression. One major question is why IKACh is preferentially expressed in pacing tissues and atria, but not ventricle. If we can understand the details of expression, assembly, and regulation of this channel, it may someday be possible to selectively express and activate it in order to terminate firing of excitable cells in arrhythmias.

DESCRIPTION (provided by applicant): Ion channels govern the activity of the thinking brain, beating heart, contracting muscle, and every cell of the body. They are targets of many therapeutic agents; mutations of ion channel genes are the cause of dozens of inherited diseases, including cardiac arrhythmias and neurological illnesses. Many of these ion channels are opened in response to changes in voltage across the cell membrane. The central long-term aim of this proposal is to understand the molecular mechanism of voltage gating of ion channels. We recently discovered an ion channel in bacteria (NaChBac) that has many of the properties of an important class of ion channels in humans: it is selectively permeant to sodium, opened (gated) by changes in membrane voltage, and inactivated in a time-dependant manner after voltage-dependant gating.NaChBac is unique in being the only voltage-dependent ion channel that can be expressed and studied in a mammalian cell line. This is important because bacterial channels are the most likely sources of sufficient protein for high-resolution structural studies (X-ray crystallography). The NaChBac protein has been crystallized and is likely to yield high-resolution structural data. It is thus crucial to investigate the structure and function of this ion channel through a combination of mutagenic and electrophysiologic studies. Information gained about its ion selectivity, voltage gating, and inactivation can then be directly correlated to the structure when obtained. Understanding this relatively simple ion channel will help us understand how the larger class of channels function, and eventually how we might target them with therapeutic agents.

DESCRIPTION (provided by applicant): The fusion of sperm and egg lead to the combination of the father's and mother's genetic information to create a new individual. In humans, as in all mammals, sperm must reach the egg, penetrate its protective coat, fuse with the oocyte membrane, and deliver its genetic material. Ion channels in the sperm mediate many of these steps, but most of these ion channels are also present in other tissues, including brain and heart. We have identified a class of ion channels, called CatSpers (Cation channel of Sperm) that are in mature sperm but not in any other tissues of the body, including developing organs. CatSpers are six transmembrane-spanning ion channel proteins localized primarily to the tail of mature sperm (Ren et al., 2001). CatSper1, the first of these ion channels to be identified, triggers calcium influx into the principal piece of the sperm tail. Calcium is required for sperm motility, and sperm from mice genetically engineered to lack the functional CatSper1 gene show a significant reduction in motility. Male mice homozygous for null-mutations in the CatSper1 gene are infertile (100%), but otherwise are completely normal. Another related sperm-specific protein, CatSper2, is also localized to its tail. Since these ion channels are only in mature sperm and required for fertility, a specific agent that blocks channel function should prevent fertilization. Experiments are proposed that will enable the expression of these novel genes in heterologous cell lines suitable for large-scale screening for small molecule blockers. This will provide a viable assay for screening for male contraceptive agents.

[unreadable] DESCRIPTION (provided by applicant): The fusion of sperm and egg lead to the combination of the father's and mother's genetic information to create a new individual. In humans, as in all mammals, sperm must reach the egg, penetrate its protective coat, fuse with the oocyte membrane, and deliver its genetic material. Ion channels in the sperm mediate many of these steps. We have identified four ion channels that are in sperm but not other tissues. CatSpers (Cation channel of Sperm) are six transmembrane-spanning ion channel proteins localized primarily to the tail of mature sperm (Ren et al, 2001). CatSper1 is an ion channel that triggers calcium-influx into the principal piece of the sperm tail. Male mice homozygous for null-mutations in the CatSper1 gene are 100% infertile, but otherwise are completely normal. Sperm from mice genetically engineered to lack the functional CatSper1 gene show a significant reduction in motility and are incapable of penetrating through the outer coat of an egg. [unreadable] We have identified two new sperm-specific proteins, CatSper3 and CatSper4 that are localized to the sperm tail. Experiments are proposed that will delineate the function of these novel ion channels and reveal their role in sperm function. Understanding these proteins will increase our understanding of fertilization and shed light on both some causes of male infertility as well as provide new targets for contraceptive agents. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The fusion of sperm and egg lead to the combination of the father's and mother's genetic information to create a new individual. In humans, as in all mammals, sperm must reach the egg, penetrate its protective coat, fuse with the oocyte membrane, and deliver its genetic material. Ion channels in the sperm mediate many of these steps, but most of these ion channels are also present in other tissues, including brain and heart. We have identified a class of ion channels, called CatSpers (Cation channel of Sperm) that are in mature sperm but not in any other tissues of the body, including developing organs. CatSpers are six transmembrane-spanning ion channel proteins localized primarily to the tail of mature sperm (Ren et al., 2001). CatSper1, the first of these ion channels to be identified, triggers calcium influx into the principal piece of the sperm tail. Calcium is required for sperm motility, and sperm from mice genetically engineered to lack the functional CatSper1 gene show a significant reduction in motility. Male mice homozygous for null-mutations in the CatSper1 gene are infertile (100%), but otherwise are completely normal. Another related sperm-specific protein, CatSper2, is also localized to its tail. Since these ion channels are only in mature sperm and required for fertility, a specific agent that blocks channel function should prevent fertilization. Experiments are proposed that will enable the expression of these novel genes in heterologous cell lines suitable for large-scale screening for small molecule blockers. This will provide a viable assay for screening for male contraceptive agents.

DESCRIPTION (provided by applicant): The fusion of sperm and egg lead to the combination of the father's and mother's genetic information to create a new individual. In humans, as in all mammals, sperm must reach the egg, penetrate its protective coat, fuse with the oocyte membrane, and deliver its genetic material. We have identified ion channels that are in spermatozoa, but not other tissues. CatSpers1-4 (Cation channel of Sperm) are 4 distinct genes present in all mammals, including humans, that encode 6 transmembrane-spanning ion channel protein subunits. These proteins form a sperm-specific, alkaline-activated, calcium-selective ion channel localized exclusively to the principal piece of the sperm tail. Male mice homozygous for null-mutations of any of the 4 CatSper genes are infertile but otherwise are completely normal. CatSper null sperm are unable to acquire hyperactivated motility and have reduced sperm endurance. Female mice lacking these genes appear completely normal, and have normal fertility. The CatSper protein spans the plasma membrane of the spermatozoan tail, and thus its extracellular surface is accessible to small molecules. The purpose of this proposal is to develop high throughput, and secondary verification screens, to identify drugs that block CatSper activity. A CatSper blocker should readily access binding sites on the external surface of sperm and thus prevent fertilization. Since CatSper is only expressed in mature sperm, a specific blocker should have no side effects on other cells and tissues, testicular function, or sperm development. PUBLIC RELEVANCE: We aim to develop an effective and safe male contraceptive. The drug will be targeted against a unique protein on the surface of mature sperm cells, called CatSper. CatSper is required for male fertility. Since the protein is only present on mature sperm, a drug inhibiting this molecule should have no effect on normal testicular or sperm development, and no effect on other organs within males or females.

DESCRIPTION (provided by applicant): The Canonical Transient Receptor Potential family of ion channel subunits comprise 7 distinct gene products (TRPC1-7), most of which are expressed in the central nervous system. Three subfamily members of this class (TRPC1, TRPC4, and TRPC5) are highly expressed in the hippocampus. These ion channel subunits forms homomeric and heteromeric channels with distinct characteristics. In addition, the currents mediated by these proteins are activated or potentiated by phospholipase C via subtypes of G protein-coupled receptors and tyrosine kinase receptors. In particular, Gq/11-linked receptors such as the type 1 metabotropic glutamate and M1, M3, and M5 muscarinic receptors, activate these currents by altering plasma membrane PIP2 and increasing intracellular Ca2+ concentrations. The function of the TRPC1/4/5 subfamily in hippocampus is not known. We hypothesize that hippocampal function is modulated by receptors that activate or potentiate these excitatory ion channels. We have generated mice lacking the TRPC4, TRPC5, both TRPC4 and TRPC5 genes, obtained the TRPC1 knockout mouse, and are breeding TRPC1/4/5 triple knockout mice. Here we propose to use these mice to understand the function of these ion channels in the hippocampus using protein localization, behavioral studies, acute brain slice recordings of hippocampus, and recordings of isolated hippocampal neurons. The results of these studies should clarify receptor-mediated alteration of hippocampal-mediated spatial and contextual memory, and identify new therapeutic targets for diseases and surgeries that affect the hippocampus. PUBLIC HEALTH RELEVANCE: Long term memory and spatial navigation rely upon the function of the hippocampus, a large region of the brain located beneath the cortical surface. Damage to the hippocampus, from oxygen starvation, encephalitis, medial temporal lobe epilepsy, tumors, and Alzheimer's disease, may result in amnesia, the inability to form or retain new memories. The basic functional unit of the hippocampus (and brain) is the richly modifiable synapse, the point where one neuron passes information to another. Synapses are endowed with a dense array of proteins that enable a panoply of modulatory influences. Foremost among the proteins that mediate synaptic function are ion channels. The function of these proteins is to initiate and control information flow from one synapse to another, and in particular, to initiate changes in intracellular calcium that control neurotransmitter release. The ion channels that are absolutely required for synaptic function in the hippocampus are largely known. Less well known are the functions of modulatory ion channels, known as TRP channels. These ion channels are activated by special classes of receptors and allow calcium to flow directly into neurons. In this proposal, we outline how we will determine the function of the canonical subfamily of TRPC channel subunits comprised of TRPC1, TRPC4, and TRPC5. Currently there are no pharmacological agents that specifically block these channels. We have genetically modified mice that lack each of these genes, as well as mice lacking combinations of all 3 of the genes. We will conduct experiments with these genetically modified mice in order to understand changes in function of the hippocampus, as determined by behavioral studies and detailed electrophysiological studies of the neurons normally expressing these channels. The long term practical benefit of such studies is to better understand how the hippocampus works, as well as define potentially new therapeutic targets that may be used to ameliorate conditions such as learning and memory deficits that occur in development, oxygen starvation during birth, central nervous system surgeries and cancers, and Alzheimer's disease.

This project is designed to study the specific function of TRPV3 in generating peripheral itch and pain sensations and determine whether targeted delivery of impermeant ion channel blockers can reduce or block these unpleasant sensations. The strategy is based on the success by our collaborators of delivering the charged sodium channel blocker, QX-314, via TRPVl into pain sensing C fibers. TRPV3, like TRPVl, is a large pore channel that is permeant to large molecules. TRPV3 is unique in that it opens at temperatures near skin temperatures (31 oC) and continues to conduct ions well above core body temperatures into the noxious temperature range. Even more surprisingly, TRPV3 continues to increase with repeated stimulation (sensitizes) unlike all other TRP ion channels, which desensitize to repeated stimuli. One ofthe striking properties of TRPV3 is its abundance in skin cells (keratinocytes) where it is much more common than other TRP channels. Recent work with a constitutively active TRPV3 channel in transgenic mice suggests that TRPV3 induces profound itch. TRPV3 is also present in peripheral sensory neurons, but its function in such neurons is not understood. Phannaceutical companies have also presented evidence that TRPV3 blockers are effective in reducing painful stimuli. We will start by defining the detailed distributions of TRPV3 in skin and neurons innervating skin, and determining whether keratinocyte TRPV3 activation produces nociceptive or pruritic mediators, such as ATP and interieukins. Using genetically modified mice, which lack all TRPV3, lack TRPV3 only in keratinocytes, or lack TRPV3 only in dorsal root ganglion neurons, we will tease apart the contributions of keratinocyte and DRG TRPV3 to pain and itch behaviors in mice. Finally, we will collaborate with the other project investigators to use the genetic, pharmacological, and channel blocking agents to selectively eliminate signals from TRPV3-expressing keratinocytes and neurons. This will be accomplished by using the large pore properties of TRPV3 to introduce blocking compounds into these cells. The overall goals are to define the peripheral circuitry of pain and itch, but also to investigate a new and exciting method of drug delivery into specific cell types that will alleviate painful and pruritic sensation. RELEVANCE (See instnjctions): Pain is familiar to all of us as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Pain can be beneficial in that it helps us avoid harmful stimuli, but can also be debilitating, significantiy interfering with a person's quality of life and function. Here we propose new targets for the treatment of pain and itch, and novel ways to silence them when they become debilitating.

DESCRIPTION (provided by applicant): The goal of the Research Training Program in Pediatric Cardiology is to attract and train highly qualified physician scientists and PhD scientists dedicated to advancing the understanding, diagnosis, treatment, and prevention of pediatric and congenital heart disease. We are requesting 8 trainee positions, on average including 4 with MD or MD-PhD degrees and 4 with PhD degrees. We will expose trainees to the clinical, translational, or basic research techniques that represent the current state of the art. We will also teach trainees the importance of teamwork, the clear communication of research findings, responsible conduct of human and laboratory research, and mentoring skills. A Research Training Executive Committee (RTEC), comprising the Program Directors and five current mentors, will oversee the progress of fellow training, career development, and mentoring, approve new mentors, and ensure that existing mentors meet the metrics of the training program. A Core Curriculum includes Work-in-Progress meetings, a T32 journal club, a cardiovascular seminar series, a course in statistical methods, a hands-on didactic series in cardiac anatomy, a career development seminar, and attendance at the Circulation Editorial Board meetings. Trainees will also participate in an annual retreat and quarterly informal meetings of all T32 trainees and mentors. Through these interchanges, clinically oriented trainees will gain understanding of fundamental cardiovascular biology, and basic science trainees will gain understanding of important clinical problems. An External Advisory Committee (EAC) will have three visits in the five year grant period and will meet with Program directors, mentors and trainees, as well as provide the RTEC with written reports of their findings. An Internal Advisory Committee will comprise senior scientists and experienced mentors who will facilitate effectiveness of training by suggesting strategies that have proved successful in training programs in other disciplines within our institution. Milestones and metrics for trainee progress and the training program itself are outlined and will be overseen by the RTEC. Considerable institutional support includes supplementation of trainee stipends and travel funds, and support for the EAC and program activities. Excellent training in rigorous experimental bench research and in translational and clinical investigation within an interactive and vibrant program should prepare graduates to become leaders in pediatric cardiology and cardiovascular research.

The objective of the Administrative Core is to allow the principal investigators to focus their energies on the scientific aspects of the study plan. Recognizing that the PI is ulfimately responsible for all aspects of the award, it is the aim of the administrafive core to handle all non-scientific tasks that pertain to the conduct of this research. To facilitate communicafion, coordinafion and planning among the projects and the PI, Dr. Clapham will be assisted by a Laboratory/scientific-program Administrator. The PI and the Administrator will meet weekly to ensure that the PI is fully informed and an acfive participant in the process of Administrafion. The Administrafive Core has three specific aims: Aim 1 is to assist the PI and his colleagues with fiscal management ofthe award. This includes processing award nofices from the NIH, managing project budgets, and assisting in the preparation of annual progress reports to the NIH. The Children's Hospital Office of Sponsored Programs (OSP) is staffed with grants management specialists who concentrate on pre-award aspects of research awards. Post-award oversight is the joint responsibility ofthe OSP and the Office of Research Finance (RFO). The Laboratory/scientific- program Administrator is responsible for daily pre- and post-award activities. Aim 2 is to provide clerical support to the PI and his colleagues for tracking annual renewals of animal and human protocols, manuscript preparafion, traveling and scheduling as it pertains to the program. Aim 3 is to assist the PI and his colleagues in ensuring effecfive communicafion, coordinafion and planning for the program. The laboratory/scientific-program Administrator will schedule and facilitate monthly research update and planning meetings to be attended by each project/core leader and the PI. The intemal advisory board (Corey, Yellen, Ji) will be invited on a quarteriy basis to attend the research update meeting, and external advisors (Julius, Caterina, Basbaum) will be invited to attend yeariy, to provide their fresh perspectives and guidance. RELEVANCE (See instnjctions): An administrafive core is necessary to the smooth function of oversight and administrafion of the acfivities of the projects.

DESCRIPTION (provided by applicant): The fundamental goal of this revised PO1 proposal is to understand how pain and itch are generated by different nociceptor and pruriceptors sensory neurons and to develop techniques that can pharmacologically silence the signals responsible, as a potential novel therapeutic strategy. The program is now entirely focused on the peripheral nervous system and on the Transient Receptor Potential (TRP) TRPV1, TRPA1 and TRPV3 channels and P2X purinergic ligand-gated ion channels, both because they are key elements in the processing of sensory signals, and even more so because they are all large pore channels allowing permeation of drug molecules into the interior of nerve cells to block excitation and transmitter release. Clifford Woolf in Project 1 will identify the function of the different subsets of TRPV1l, TRPV3, TRPA1 and P2X3 expressing primary sensory neurons in pain and itch by transiently silencing their axons after delivery of the permanently charged sodium channel blocker QX-314 through the channels. Bruce Bean in Project 2 will also explore how permeation of drugs through TRP and purinergic ligand-gated ion channels can be used to silence primary sensory neurons, but by using delivery of cationic calcium channel blockers to disrupt vesicle release in the periphery to reduce neurogenic inflammation and in the spinal cord to eliminate synaptic transmission. David Clapham's Project 3 will identify how and where TRPV3 contributes to pain and itch (in keratinocytes or sensory neurons), an important issue since TRPV3 antagonists are analgesic in preclinical models and are about to be tested clinically, and will also explore if permeation of ion channel blockers through TRPV3 can be used to modify the contribution of keratinocytes and primary sensory neurons to pain and itch. Qiufu Ma in Project 4 will use genetic techniques to silence defined primary sensory neurons to tease out their specific role in pain and itch. A primary sensory neuron sp

DESCRIPTION (provided by applicant): The fusion of sperm and egg lead to the combination of the father's and mother's genetic information to create a new individual. In humans, as in all mammals, sperm must reach the egg, penetrate its protective coat, fuse with the oocyte membrane, and deliver its genetic material. We have identified ion channels that are in spermatozoa, but not other tissues. CatSpers1-4 (Cation channel of Sperm) are 4 distinct genes present in all mammals, including humans, that encode 6 transmembrane-spanning ion channel protein subunits. These proteins form a sperm-specific, alkaline-activated, calcium-selective ion channel localized exclusively to the principal piece of the sperm tail. Male mice homozygous for null-mutations of any of the 4 CatSper genes are infertile but otherwise are completely normal. CatSper null sperm are unable to acquire hyperactivated motility and have reduced sperm endurance. Female mice lacking these genes appear completely normal, and have normal fertility. The CatSper protein spans the plasma membrane of the spermatozoan tail, and thus its extracellular surface is accessible to small molecules. The purpose of this proposal is to develop high throughput, and secondary verification screens, to identify drugs that block CatSper activity. A CatSper blocker should readily access binding sites on the external surface of sperm and thus prevent fertilization. Since CatSper is only expressed in mature sperm, a specific blocker should have no side effects on other cells and tissues, testicular function, or sperm development. PUBLIC RELEVANCE: We aim to develop an effective and safe male contraceptive. The drug will be targeted against a unique protein on the surface of mature sperm cells, called CatSper. CatSper is required for male fertility. Since the protein is only present on mature sperm, a drug inhibiting this molecule should have no effect on normal testicular or sperm development, and no effect on other organs within males or females.

? DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease is caused by mutations in the Pkd1 (85%) or Pkd2 (15%) genes. PKD morbidity is associated with primarily renal, but also liver, pancreatic, and vascular abnormalities such as intracranial aneurysms and aortic artery weakness. There are no effective therapies at present. Primary cilia house important regulators of cell growth and division. Defects in genes regulating cilia formation and function result in serious lifelong and adult-onset diseases. Here we focus on polycystins, the genes mutant in polycystic kidney diseases that are among the most common inherited disorders. The organizing principal of this proposal is that ciliary polycystin channels modify ciliary calcium levels that in turn modify ciliary signaling pathways regulating growth and cell division, and ultimately cyst formation. We measured ciliary calcium concentration and identified ion channel currents (Icilium) in single cilia. We found that calcium changes are largel confined to the cilium. We directly measured primary cilia currents under whole-cilium voltage clamp and found that the basal current was mediated by PC1-L1/PC2-L1 heteromers. These channels rapidly and dramatically alter cilia calcium levels. We were able to conduct these experiments by generating fluorescent indicators for calcium measurements that specifically target cilia. We showed that ciliary calcium levels regulate Smoothened-activated Gli1 transcription and ciliary tip accumulation of Gli2. We are crossing Pkd-mutant mice with these indicator mice. We focus on the function of the polycystin proteins themselves rather than downstream signal transduction pathways. Here we propose to measure ion channel currents in primary cilia from freshly isolated kidney collecting duct tubules, and tubular cells in intact tubules. The overall goal is to determine whether PC1 and PC2, the proteins mutant in polycystic kidney disease, also function as an ion channel complex in cilia. Our hypothesis is that PC-L1 proteins regulate cilia calcium to high levels and as cells mature and differentiate, they express PC proteins and reduce cilia calcium. In the experiments proposed, we will first identify the type of channel, PC or PC-L1, that is present in embryonic, neonatal, young adult, and mature adult mice. This requires careful evaluation of PC and PC-L1 biophysical and pharmacological properties. We will then determine and compare the domains of PC and PC-L1 that enable them to regulate calcium in cilia in distinct ways. Finally, we will determine how PC1, the more commonly affected protein in polycystic kidney disease, regulates the pore of the PC2-forming channel.