Bruce P. Bean - US grants

The rate and force of the heart's condition are regulated by the autonomic nervous system and by circulating hormones. A primary mechanism of regulation is modulation of the number and behavior of ionic channels in cardiac cell membranes. The goal of the proposed research is to use newly available methods of ionic channel biophysics to understand how the operation of cardiac channels is changed by hormonal stimulation. With a primary emphasis on regulation of the channels carrying slow inward calcium current, the experimental approach will combine patch clamp recordings of currents through single calcium channels with gigaseal electrode recordings of currents from whole cells, using fluctuation analysis techniques to estimate changes in the number of functional calcium channels in a cell. The ability to dialyze the inside of a single cell will permit investigation of the possible role of internal calcium as a second messenger. These approaches will help provide answers to basic questions about calcium channel regulation. Do Beta-adrenergic agonists increase calcium current by recruiting an entirely new population of channels or by modifying the operation of a single class of channels? How are calcium channel kinetics, voltage-dependence, and sensitivity to channel blockers changed by adrenergic stimulation? Does acetylcholine regulate calcium channels by a direct or a second messenger action? Are changes in internal calcium important for acetylcholine's action? How does angiotensin's regulation of the calcium channel differ from that of adrenaline? Does adenosine decrease calcium current by a common mechanism with acetylcholine? Two specific questions about potassium channel regulation will also be studied. In adrenergic increases of voltage-activated potassium channels, are new channels recruited or old channels modified? Does adenosine induce resting potassium current by a second messenger system? Neurotransmitter and hormonal regulation of calcium and potassium channels is important not only for the normal physiological control of the heart, but also in the genesis of some arrhythmias. The proposed experiments may also shed light on related physiological control systems in exocrine and endocrine cells, smooth muscle, and neurons.

Many neurotransmitters act by opening or closing ionic channels in the membranes of target cells. Transmitter control of channels is a fundamental mechanism underlying the regulation of the heart and other organs by the nervous system, as well as for synaptic transmission between nerve cells. The mechanisms by which transmitters control channels are poorly understood; in most cases, the link between transmitter binding and channel control seems less direct than for the end-plate acetylcholine receptor, where the channel is tightly coupled to the receptor. The long term-goal of the proposed research is to use an electrophysiological approach to understand the range of mechanisms by which transmitters control the operation of ionic channels. Of special interest are cases in which transmitter- channel coupling may be indirect, including transmitter modulation of voltage-dependent channels. Transmitter control of channels will be investigated in heart muscle, smooth muscle, and neurons from frogs, rats, and rabbits. Patch clamp techniques will be used to record ionic currents, both at the level of the whole cell and the single channel, using single cells dispersed from tissue or grown in culture. The approach will help answer basic questions about several related transmitter mechanisms. Do beta-adrenergic agonists increase cardiac calcium current by shifting the voltage- dependence of the channels? Is alpha-adrenergic depression of calcium current in sensory neurons due to a change in the voltage- dependent operation of the channels or to elimination of a fraction of the channels? What channels underlie the hyperpolarization of nerve cells produced by norepinephrine? What channels does external ATP open to produce excitation in heart cells, smooth muscle cells, and neurons? What channels in central neurons are controlled by glutamate, acetylcholine, and norepinephrine? Neurotransmitter control of ionic channels is a basic process for the normal operation of the brain, the heart, and the vascular system. Understanding the mechanisms involved will help understand pathological states such as cardiac arrhythmias, hypertension, epilepsy, depression, and chronic pain.

Neurons in the mammalian brain possess a rich variety of voltage-dependent ion channels, but there has been little detailed analysis of how particular ion channels work together to regulate the firing patterns of mammalian central neurons. In part, this has been due to limitations in voltage-clamping central neurons, especially for studying the large voltage-activated currents that flow during the action potential. The goal of the proposed research is to understand how the distinctive firing properties of cerebellar Purkinge neurons are produced by particular ion channels. The work is based on using a preparation of dissociated Purkinje neurons that allows a high-quality voltage-clamp of voltage- activated currents. Preliminary data show that the dissociated cells retain two of the distinctive firing properties of Purkinje cells in vivo, spontaneous firing and formation of complex action potentials. The experimental design will combine current clamp recordings of action potential firing with a voltage-clamp analysis of the voltage-dependent sodium, potassium, and calcium channels that underlie the action potentials. Voltage clamp experiments will use ionic substitution and specific channel blockers, especially peptide toxins, to distinguish the contributions of particular channel types to the overall sodium, calcium, and potassium currents. Action potential waveforms will be used as command voltages to determine the contribution of particular ion channels to firing patterns. A particular focus will be to characterize a novel repolarization-gated sodium current using single channel and whole-cell recordings, and to understand the role of the current in spontaneous firing and in the formation of multi-spike action potentials. Understanding the mechanisms involved in regulating the electrical excitability of central neurons will help in understanding the normal function of the nervous system as well as pathophysiological states resulting form stroke, intoxication, and epilepsy.

Acetylcholine acting through muscarinic receptors modulates the excitability of neurons by altering various sodium, calcium, and potassium channels. This modulation of excitability plays a central role in cholinergic regulation of wakefulness, cognition, and memory. The goal of the proposed research is to understand muscarinic changes in action potential formation and firing patterns in terms of modulation of particular ion channels. Patch clamp techniques will be used to study the control of sodium, potassium, calcium channels in sympathetic, hippocampal, and dopamine neurons by muscarinic stimulation. The primary experimental preparation will be acutely isolated neurons, which allow voltage-clamp with sufficient speed to characterize modulation of the large, rapid currents flowing during action potentials. This will extend previous work using brain slices, in which only relatively small and slow currents could be studied under voltage-clamp. We will directly examine currents flowing during action potentials by first recording action potential wave-forms and, in the same cell, using those wave forms as command voltages in voltage clamp experiments. By using pharmacological tools to separate individual current sand performing both current-clamp and voltage-clamp experiments in the presence of acetylcholine, it should be possible to determine the role of modulation of particular channel types in contribution to overall changes in excitability. Muscarinic control of ion channels and neuronal excitability is a basic process for the normal operation of the brain, and evidence suggests changes in the aging brain. Changes in muscarinic modulation of hippocampal neurons may be centrally involved in the symptoms of Alzheimer's disease, and better understanding of the channels involved may lead to novel therapeutic interventions, for example by using ion channel blockers to mimic effects of acetylcholine.

Four investigators with long standing interest in ion channels and synaptic biology have come together to study effects of acetylcholine (ACh) on the activation and modulation of ion channels in the central nervous system. Changes in the effective concentration of ACh or in the number of ACh receptors (AChRs) have been associated with cognitive disorders, attention deficits, memory loss in normal subjects and in Alzheimer's patients, in Parkinson's Disease and in drug seeking behaviors. Work described in this Program Project includes analyses of nicotinic and muscarinic mechanisms that may shed light on these disorders. The Program developed naturally out of collaborative projects already begun by the four investigators. Together, we plan to investigate the regulation of nicotinic AChRs by neuregulins, a family of trophic factors expressed in the brain; the role of nicotinic and muscarinic receptors in modulating synaptic transmission; the modulation by ACh of several types of voltage gated ion channels; the distribution and role of G-protein coupled potassium channels (GIRKs) in regulating neuronal firing. Several different regions of the brain will be studied including the hippocampus, substantia nigra, ventral tegmental area, globus pallidus, medial habenula, interpeduncular nucleus, and cerebellum. Several preparations will be employed including freshly dissociated neurons, nerve cell cultures, brain slices and genetically altered mice. The primary tools are electrophysiological as we are primarily concerned with ACh induced changes in synaptic function and neuronal firing patterns. The projects are closely related and interactions have already emerged that greatly facilitate progress in each area. These interactions will broaden the scope of our work in the future.

DESCRIPTION (provided by applicant): Work over the last twenty years has identified a remarkable number of proteins that form ion channels in the mammalian brain. In many cases, we have detailed information about the molecular characteristics of the channels and on how they can by modulated by G proteins and second messengers. However, we understand much less about how the many voltage-dependent channels expressed in a single central neuron work together to produce the firing patterns characteristic of that particular neuron. The goal of the proposed research is to understand how the firing properties of cerebellar Purkinje neurons are produced by particular combinations of ion channels. The work will combine current clamp recordings of action potential firing with a voltage-clamp analysis of the voltage-dependent sodium, potassium, and calcium channels underlying the action potentials. Studies on Purkinje neurons in cerebellar slices will examine ion channels and intrinsic membrane properties of dendrites and soma and will investigate how these interact under physiological conditions. The electrophysiological characterization will be complemented by immunocytochemical localization of particular sodium, calcium, and potassium channels. A preparation of dissociated cell bodies will allow a high-quality voltage-clamp of voltage-activated currents. Ionic substitution and specific channel blockers, especially peptide toxins, will be used to distinguish the contributions of particular channels. Action potential waveforms will be used as command voltages to determine the contribution of particular ion channels to particular firing patterns, with a special focus on understanding pacemaking activity and complex spikes. Understanding the mechanisms involved in regulating the electrical excitability of central neurons will help in understanding the normal function of the nervous system as well as pathophysiological states resulting from stroke, intoxication, and epilepsy.

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) in the ventral hypothalamus contains the master clock of the brain, controlling the 24-hour circadian rhythm of physiological functions. Recent work has identified in SCN neurons many components of a molecular clock, composed of transcription factors and kinases that interact in regulatory feedback pathways. A major unanswered question is how the molecular clock is connected to the electrical activity of the neurons. The goal of the proposed research is to understand the ionic conductances that control spontaneous firing of SCN neurons and the circadian variation in their firing rate. We will do complementary experiments on intact SCN neurons in brain slice and on a preparation of acutely-dissociated neurons in which pacemaking is maintained. The brain slice preparation allows recordings under the most undisturbed conditions, while dissociated neurons enable voltage-clamp recordings with high time- and voltage-resolution and permit fast solution exchanges and readily reversible application of drugs and channel blockers. Electrophysiological and pharmacological identification of channels underlying various components of electrical current will be complemented by immunocytochemical identification of the channels expressed in the neurons. In addition to characterizing the ionic currents that directly drive pacemaking of SCN neurons, we will characterize other currents that are important for regulating its frequency. We will then determine which of these currents change in amplitude, voltage-dependence, or kinetics to produce the diurnal variation in firing frequency. Finally, we will attempt to discover how the molecular clock is linked to electrical pacemaking and determine whether the electrical clock is regulated by alteration in the activity of kinases or other second messenger pathways. Understanding the mechanisms involved in regulating the excitability of SCN neurons will help in understanding the normal function of the nervous system as well as dysregulation of sleep, attention, and hormonal release.

Project Summary/ Abstract Pain is signaled by generation of action potentials in a specific population of primary sensory neurons known as nociceptors. The most effective form of pain relief without loss of consciousness is provided by administration of local anesthetics, which act by inhibiting voltage-dependent sodium channels and thereby depressing electrical excitability. Clinically-used local anesthetics are molecules that exist at least partially in a hydrophobic, uncharged form that can enter neurons through the cell membrane. These anesthetics enter and inhibit excitability in all neurons, not just nociceptors, and thus can have many undesirable effects (including paralysis and block of autonomic signaling) in addition to blocking pain. The proposed research is based on a recent finding that sodium channel blocking drugs can be targeted selectively to nociceptors by co-applying a permanently charged derivative of lidocaine (QX- 314) with capsaicin, an agonist for TRPV1 channels. The underlying hypothesis, supported by the preliminary data in the proposal, is that QX-314 can enter nociceptors by passing through the pore formed by TRPV1 channels. The overall goal of the proposed research is to identify combinations of TRPV1 activators and charged sodium channel blockers that optimize the block of excitability of nociceptive sensory neurons. Specific questions to be addressed include: What is the size limit for effective entry of charged sodium channel blockers? How does the time course of blocker entry depend on the nature and concentration of the TRPV1 agonist? Can blocker entry and accumulation be enhanced by activation of protein kinase C? Are there TRPV1 agonists that allow QX-314 entry without first stimulating firing of action potentials? What is the relative potency of intracellular QX-314 for blocking the different types of sodium channels known to be important for excitability of nociceptors? These questions will be addressed using patch clamp experiments on native TRPV1 channels and sodium channels in rat dorsal root ganglion neurons, with additional experiments using heterologous expression of cloned TRPV1 channels. Characterizing these mechanisms should facilitate the development of new clinical treatments for pain relief based on the targeted entry of charged sodium channel blockers into pain-sensing neurons. Such treatments should be highly advantageous for more selective pain relief in childbirth, surgery, and dental procedures and possibly for some forms of chronic neurogenic pain.

DESCRIPTION (provided by applicant): Work over the last twenty-five years has identified a remarkable number of proteins that form ion channels in the mammalian brain. In many cases, we now have detailed information about the molecular characteristics of the channels. However, we understand much less about how the many types of ion channels present in a single central neuron work together to produce the firing patterns characteristic of that particular neuron. The goal of the proposed research is to understand how the firing properties of particular central neurons are produced by particular combinations of ion channels. The work will combine current clamp recordings of action potential firing together with voltage-clamp analysis of the ion channels regulating both subthreshold and suprathreshold electrical properties. One project will examine the channels that are important for generating and regulating spontaneous firing of midbrain dopaminergic neurons, including an exploration of differences between dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area. The role of calcium entry though different types of calcium channels will be evaluated, and we will test the hypothesis that electrogenic current from the sodium-calcium exchanger is important for pacemaking. A second project will determine the ion channels important for enabling very rapid firing in cerebellar Purkinje neurons, a model system for other fast-spiking neurons in the brain. A third project will explore how active subthreshold conductances interact with excitatory and inhibitory synaptic potentials to amplify or dampen their effects. An emerging common theme is that some conductances (e.g. voltage-dependent sodium channels and A-type potassium channels) have dual roles, generating both large transient currents determining spike shape and also very small subthreshold currents between spikes that are important for determining firing patterns. Understanding the channel gating mechanisms that integrate these two functions is a major challenge. Recordings will be made both from intact neurons in brain slices and from acutely dissociated neurons, where high-quality voltage clamp of large currents is possible. Dynamic clamp and action potential clamp techniques will link current clamp and voltage clamp recordings. Understanding the mechanisms involved in regulating the excitability of central neurons will help in understanding the normal function of the nervous system as well as pathophysiological states resulting from epilepsy, Parkinson's disease, and other disorders. PUBLIC HEALTH RELEVANCE: The goal of the research is to understand the basic mechanisms that control electrical activity of neurons in the mammalian brain. The processes being investigated are not only critical for understanding the normal function of the brain but also for understanding pathophysiological states such as epilepsy, where electrical activity is excessive and uncontrolled. A major part of the project will characterize electrical activity in midbrain dopaminergic neurons, whose death is the causative event in Parkinson's disease. Specifically, we will analyze modes of entry of calcium into dopaminergic neurons and determine what specific ion channels are important for calcium entry, which has been hypothesized to be the proximate cause of cell death in Parkinson's disease. Understanding the role of calcium entry in the function of dopaminergic neurons could suggest particular pharmacological targets for slowing or preventing cell death while maintaining electrical function of the neurons.

instrucfions): The goal ofthe proposed research is to develop methods for loading charged calcium channel blockers into sensory neurons by entry through large-pore TRP channels. Because many TRP channels are selectively expressed in pain-sensing neurons (nociceptors), calcium channel blockers that are membrane- impermeant and inactive from the outside of cells but active from the inside can be targeted to pain-sensing neurons without affecting other types of neurons. Two major functions of calcium channels in nociceptors can be targeted by this strategy: 1) Block of transmitter release from presynaptic terminals in the spinal cord, and 2) Blocking of peripheral release of neuropeptides such as CGRP and substance P involved in inflammatory pain. Charged calcium channel blockers that block N-type calcium channels from the inside but not the outside of cells will be co-applied with agonists of TRPVl, TRPV3, TRPAl, and P2X channels to target the blockers to central and peripheral terminals of primary nociceptors. Targeting of calcium channel blockers to nociceptors should be very useful as both an investigational tool and as a therapy. As a tool, it will allow highly selective silencing of synaptic transmission from specific populations of nociceptors. Making use ofthe information and tools that will be developed In the Clapham, Ma, and Woolf projects, we can use specific agonists for various large-pore channels to target calcium channel blockers to specific subsets of neurons expressing TRPVl, TRPV3, TRPAl, and P2X channels. Doing so will allow selective block of specific modalities of pain and itch. As a therapy, the strategy should allow block of pain signaling from primary noiceptors into the spinal cord without impairing non-pain circuits in the spinal cord. Applying the strategy to block release of inflammatory neuropeptides such as substance P and CGRP from peripheral C-fiber terminals should provide a way of reducing inflammatory pain in in skin and other tissue. RELEV/VNCE (See instructions): The goal of the research in the overall Program Project is to develop new clinical treatments for pain and itch. The goal ofthe research in this project is to identify combinations of charged calcium channel blockers and TRP agonists suitable for subsequent in vivo behavioral experiments in rats and ultimately in humans.

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

The goal of the proposed research is to take a biophysical approach to understand how antiepileptic drugs targeted to voltage-dependent sodium channels regulate neuronal firing by differentially binding to different gating states of the channels. The work brings together two lines of research in the laboratory, one characterizing the state-dependent interaction of drugs like lidocaine, phenytoin, carbamazepine, and lacosamide with sodium channels and the other exploring how gating of sodium channels regulates firing of a variety of mammalian central neurons. A key property of antiepileptic drugs is differential binding to different gating states of sodium channels, but how this changes firing of particular kinds of central neurons to control pathological neuronal activity is poorly understood. For example, higher affinity binding to open and inactivated states results in use-dependence, with increased inhibition as channels cycle through open and inactivated states during action potentials. Yet, this does not easily explain their clinical action, because use-dependence might predict more potent inhibition of GABAergic inhibitory neurons, which typically fire at high frequencies, than glutamatergic excitatory neurons, which typically fire more slowly. We will examine how antiepileptic drugs interact with the gating of neuronal sodium channels and explore how state-dependent binding and unbinding regulates the firing patterns of a variety of excitatory and inhibitory neurons. We will follow up preliminary data showing that carbamazepine, phenytoin, and lamotrigine are all more effective in inhibiting firing of slower-firing glutamatergic pyramidal neurons than fast-spiking GABAergic neurons. We will analyze how these drugs and others (including the new anti- epileptic cannabidiol and a novel, more potent carbamazepine derivative) interact with gating of both native and cloned sodium channels and how the resulting changes in sodium current modify the firing patterns of a variety of excitatory and inhibitory neurons in a manner depending on the repertoire of other channels. The experimental design will combine recordings of action potential firing with voltage-clamp analysis of the underlying sodium currents, using intact neurons in brain slice, acutely dissociated neurons, and heterologously expressed cloned channels. A key feature will be to study action potential firing, channel gating kinetics, and drug action at 37 °C.