Stephen W. Jones - US grants

This project will characterize peptidergic transmission in bullfrog sympathetic ganglia, as a model system for the slow, modulatory action of peptides in the nervous system. The actions of peptides, particularly T-LHRH (the luteinizing hormone-releasing hormone of the teleost fish) and substance P, will be studied on individual cells of the ganglia under voltage clamp. The inhibitory action of peptides on the M-current, a novel voltage-dependent potassium current, will be investigated. Synaptic potentials resulting from the action of peptides, including the late, slow EPSP, will be studied. Other peptides, which may be present in the ganglia, will be tested for pre- or postsynaptic effects, and for modulatory effects on the acetylcholine receptors of ganglion cells. The chemical nature of the peptides present in the ganglion will be determined by high performance liquid chromatography, and possibly amino acid analysis and sequencing. Enzymatic degradation of peptides, and its possible role in the physiological action of the peptides, will be examined. The receptors for T-LHRH and substance P will be identified by in vitro binding studies, and will be located in the ganglia by autoradiography. This multidisciplinary approach should increase our understanding of the fundamental mechanisms involved in the generation of peptidergic synaptic actions in the nervous system.

The long term goal of this work is to understand the mechanisms that generate neuronal activity, and modulation of that activity by neurotransmitters. The first step is characterization of the voltage- dependent ion channels of frog sympathetic neurons, using whole-cell and single channel patch clamp techniques. These cells are particularly favorable for voltage clamp studies, and much is already known about the channels underlying electrical activity in sympathetic neurons. A major part of the project will involve study of the voltage-dependent calcium channels, and comparison of those channels to calcium channels in other cell types. Kinetic models of the behavior of calcium and other channels will be developed, to construct a computer model of electrical activity in these cells. The model will be tested by comparing action potentials, and patterns of action potentials, generated by the model to those actually produced by sympathetic neurons. This analysis of the normal behavior of the cell will be supplemented by study of the effects of neurotransmitters on the preexisting voltage-dependent currents. One goal is to explain quantitatively the increased excitability of frog sympathetic neurons induced by slow synaptic potentials.

9315886 Durand This award will fund a project to investigate parameter estimation techniques for use with neurophysiological systems. The investigators will compare and test current and new parameter estimation techniques; that is, Linear Associative Memories. These techniques will be applied to the estimation of electrotonic and membrane ionic channel parameters. This proposal was submitted to the Biosystems Analysis and Control Initiative which encouraged the submission of proposals that through the study of neurophysiological systems could advance engineering science and advance the knowledge of biological systems through the use of engineering analysis techniques. The research being funded by this award has the potential to assist in the modeling of neurophysiological data and advance the field of engineering in the area of parameter estimation. ***

The long term goal of this work is to understand the mechanisms that generate neuronal activity, and modulation of that activity by neurotransmitters. The first step is characterization of the voltage- dependent ion channels of frog sympathetic neurons, using whole-cell and single channel patch clamp techniques. These cells are particularly favorable for voltage clamp studies, and much is already known about the channels underlying electrical activity in sympathetic neurons. A major part of the project will involve study of the voltage-dependent calcium channels, and comparison of those channels to calcium channels in other cell types. Kinetic models of the behavior of calcium and other channels will be developed, to construct a computer model of electrical activity in these cells. The model will be tested by comparing action potentials, and patterns of action potentials, generated by the model to those actually produced by sympathetic neurons. This analysis of the normal behavior of the cell will be supplemented by study of the effects of neurotransmitters on the preexisting voltage-dependent currents. One goal is to explain quantitatively the increased excitability of frog sympathetic neurons induced by slow synaptic potentials.

The goal of this project is to characterize voltage-dependent ion channels of neurons, and their regulation by neurotransmitters. Both frog sympathetic neurons and rat thalamic neurons will be studied, and the properties of voltage-dependent calcium channels will be emphasized. Whole-cell and single-channel patch damp methods will be used. Long-term objectives of this work are to clarify how the detailed properties of specific types of ion channels contribute to the overall electrical behavior of the cell, and to the cell's role in neuronal circuits and behavior. One long-term goal of the work on thalamic neurons is to determine the ionic mechanisms underlying absence seizures (petit mal epilepsy). Four specific aims relate to frog sympathetic neurons. (1) The properties of a newly recognized calcium current will be further characterized. Specific experiments include the effect of divalent cations (Ca2+ and Ba2+) on current through this channel, and the effects of cone snail and spider toxins. One goal is to determine whether this channel was previously misidentified as the basis of the whole-cell omega- conotoxin-sensitive N-current. (2) The voltage- and Ca2+-dependence of inactivation of the N-type calcium current will be examined. The increased inactivation caused by inhibition of protein phosphatases will be examined at the single-channel level. (3) Modulation of calcium channels by neurotransmitters and G proteins will be examined at the single-channel level. The biochemical mechanisms of modulation of calcium current, and of the M-type potassium current, will be compared. (4) The mechanisms of ion permeation and block of calcium channels will be characterized. A fifth specific aim relates to neurons acutely isolated from the ventrobasal nucleus of the thalamus of neonatal rats. (S) Kinetic and pharmacological criteria will be used to determine which types of high-voltage-activated calcium channels are present in thalamic neurons. The regulation of thalamic calcium channels by neurotransmitters will be investigated. These studies will contribute to our understanding of the diversity of neuronal calcium channels, and their functions in neuronal excitability.

The long range goals of this project are to characterize the basic mechanisms underlying activity of voltage-dependent ion channels, and the roles that ion channels play in the electrical activity of neurons. The specific aims for the next grant period focus on T-type calcium channels and delayed rectifier potassium channels, with emphasis on channel inactivation. Most studies will deal with biophysical characterization of these channels in a heterologous expression system, human embryonic kidney (HEK 293) cells, but experiments will also be performed on channels natively expressed in neurons (mostly, acutely isolated thalamic relay neurons). There are four specific aims: (1) Kinetics of the alpha1G T-type calcium channel. The kinetics of activation and inactivation will be examined. Specific questions include the state-dependence of inactivation and recovery, the completeness of inactivation at steady-state, and the basis of cumulative inactivation during repetitive depolarization. One goal is construction of a kinetic model, which will be used in the future both as an operational model and as a basis for structure-function studies of the molecular mechanism of channel gating. (2) Kinetics of T-currents in thalamic neurons. The properties of native T-currents will be compared qualitatively and quantitatively to alpha1G, to determine whether the kinetic model developed for alpha1G in HEK 293 cells is applicable to neuronal T-current. (3) Permeation and gating of the Kv2.1 potassium channel in low K+. The basis of the slow channel closing observed with Na+ as the permeant ion will be investigated, to determine whether it reflects effects of permeant ions on channel activation, or induction of a Na+- permeable "inactivated" state. It will be determined whether long depolarizations induce changes in ion selectivity, or "immobilization" of gating charge. (4) Inactivation of native potassium channels. The voltage-dependence of inactivation, and cumulative inactivation during repetitive pulses, will be examined for delayed rectifiers of frog sympathetic neurons and rat thalamic neurons, to test for inactivation from closed states.

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the mechanisms underlying gating of voltage-dependent channels. Voltage-dependent channels are the basis of electrical activity in neurons and other excitable cells, and many diseases involve abnormalities in ion channel behavior (e. g., epilepsy). Specifically, we will focus on two channels: T-type calcium channels, and Kv-type delayed rectifier potassium channels. We will use di- and trivalent cation blockers of T-channels as probes for the channel pore. We will investigate the voltage- and state-dependence of channel block. We will ask whether blocking ions can enter and/or exit the pore when the channel is closed, or when it is inactivated. In essence, this asks whether the gates of the channel are on the intracellular side of the selectivity filter, on the extracellular side, or both. Regarding Kv channels, we will begin to analyze the structural basis of a recently characterized form of slow inactivation, called U-type inactivation, that occurs primarily from "partially activated" closed states along the pathway for channel activation. Using the Shaker K channel (with the N-terminal region deleted to remove fast inactivation), we will examine whether mutations affect C-type inactivation, U-type inactivation, or both. We will examine mutations at two positions: T449, a site near the outer mouth of the pore that strongly affects both slow inactivation and TEA block; and P475, where (in the Kv2.1 channel) mutations greatly enhance U-type inactivation. We will also examine the effects of mutations at the corresponding sites in the Kv2.1 channel, where we have found the wild-type channel to exhibit purely U-type inactivation. These studies will primarily use whole-cell voltage-clamp recording from cloned channels expressed in a mammalian cell line, HEK 293 cells. An important feature of our analysis is the use of kinetic models of channel gating both as empirical descriptions of our results, and as working hypotheses regarding the molecular mechanisms of channel gating. We expect our results to provide important information regarding the location of the gates that regulate ion flow, for different gating processes: activation, fast inactivation (for T-channels), and two forms of slow inactivation (Kv channels).