Ann Rittenhouse - US grants

DESCRIPTION: (adapted from Applicant's Abstract) The long-term goal of the experiments described in this proposal is to identify sources of plasticity of voltage-activated calcium (Ca) channels. The N-type Ca channel has been found only in nerve cells and neuronally-derived tissues, is associated with the regulation of neurotransmitter synthesis and release from presynaptic nerve endings, and in turn, is modulated by many of these same neurotransmitters. N-type Ca channels are the most highly modulated Ca channel in the brain in that more pathways exist for modulation of this Ca channel than for any other, and because of this, are thought to play a special role in the brain. N-type Ca channels display endogeous, heterogeneous activity, called modes, where measured unitary conductance and kinetics vary from mode to mode. Each mode can last for seconds to minutes. At least six modes have been identified and are a source of neural plasticity at the level of the N-type Ca channel. The specific aims of this project are to 1) identify the causes of modal gating and permeation of N-type Ca currents that give rise to channel plasticity, 2) determine whether modulation of N-type Ca channel behavior by neurotransmitters and cellular signals alters the frequency of occurrence and/or the biophysical characteristics of these modes, and 3) identify differences between the subunit composition of N-type Ca channels in sympathetic neurons and in the brain and determine whether differences in the expression level of the subunits is an additional source of plasticity.

DESCRIPTION (provided by applicant): The long-term goal of this lab is to identify sources of plasticity at the level of single proteins. Our working definition of plasticity at this level is the ability of a protein's behavior to vary, not simply due to an increase or decrease in its activity as with acute activation of an enzyme, but rather by qualitative changes in its behavior. Single N-type Ca channels display heterogeneous activity, called modes, where unitary conductance and kinetics are stable for seconds to minutes before abruptly changing to a new pattern of activity. Because the transitions among modes result in qualitative changes in N-type Ca channel activity, they can be considered plastic proteins. The N-type calcium (Ca) channel is found only in nerve cells and neuronally-derived tissues. It is the most extensively modulated Ca channel in the brain in that more pathways exist for its modulation than for any other, and because of this, it is thought to play a critical role in synaptic plasticity. The proposed experiments test the hypothesis that transmitters exert their actions on N-type Ca channels by activating signaling molecules that shift channel activity from one mode to another. The specific aims of this project are the following: 1) confirm that phosphorylation of the N-type Ca channel by protein kinase C (PKC) stabilizes an inactivating mode with long openings; 2) determine whether G-proteins protect channels from inactivation; 3) determine whether arachidonic acid (AA)-induced inhibition stabilizes a null activity mode; 4) demonstrate that transmitters, use PKC and AA to modulate N-type currents. Whole cell and unitary N-type currents will be studied with standard patch clamp techniques in superior cervical ganglion neurons, a preparation rich in N-type Ca channels. The effects of signaling molecules on current amplitude, gating kinetics, and rates of transition between different modes will be analyzed quantitatively. We expect to find that transmitters do converge on these signaling molecules to modulate Ca currents by stabilizing particular modes. If true, plasticity of N-type Ca channels may be a building block for emergent forms of plasticity, observed at synapses and in neural circuits. Moreover, these results should establish new signal transduction cascades for N-type Ca channel modulation, which may also be present in central neurons. Further understanding of the modulation of N-type Ca channel modes might allow the design of new pharmaceutical agents that act to stabilize modes which alter net Ca influx. This could help minimize cytotoxicity that can occur during cerebral vasospasm, stroke, epilepsy; and reduce mobility from cognitive, and/or learning and memory disorders.

[unreadable] DESCRIPTION (provided by applicant): Inappropriate electrical activity gives rise to devastating brain disorders including, epilepsies and cell death following ischemia. These diseases trigger damaging inflammatory responses that elevate free arachidonic acid (AA) levels. Under these pathological conditions, excessive Ca2+ influx leads to cell death. By understanding the role that AA serves in modulating nerve cell excitability and survival, we hope to identify therapeutic targets that will minimize nerve cell death due to inflammatory signaling. My lab has found that AA inhibits two Ca2+ currents called L- and N-currents in superior cervical ganglion (SCG) neurons by promoting channel closing. AA also increases N-current amplitude by acting at a distinct site. As with inhibition, enhancement is mimicked by stimulating M1 muscarinic receptors and requires phospholipase A2, indicating functional relevance of AA's dual actions. We have measured increased AA release from individual SCG following muscarinic stimulation using gas chromatography (GC) and mass spectroscopy (MS). Three additional, distinct peaks with a mass/charge ratio (m/z) equal to that of AA are also released suggesting that SCGs acutely synthesize AA isoforms with novel double bond patterns. AA normally has four cis double bonds. Our findings appear to be the first study documenting AA isoforms in neurons. However a recent report described a peroxidation process in endothelial cells that generates AAs with one of the bonds in the trans- conformation (TAAs). Moreover TTAs appear to mediate oxidative stress-induced microvascular degeneration. These findings raise many questions surrounding the roles of AA versus its isoforms in normal and pathological nerve cell functioning. Because of the possible clinical relevance of AA isoforms as therapeutic targets for the treatment of ischemia, the following specific aims are proposed: Aim I. Define the chemical structures of the AA isoforms released from SCG using GC-MS and nanospray ionization MS-MS. Characterizing the changes in AA double bond geometry and/or location will allow us to identify new potential proinflammatory signaling molecules. Aim II. Separate, collect and test the four AA isoforms found in SCG neurons for their ability to modulate whole-cell Ca2+ currents of SCG neurons and recombinant channels transiently transfected into HEK293 cells. Aim III. Determine whether any of the AA isoforms alter action potential firing. Whether particular AA isoforms promote nerve cell growth or death will also be tested. These studies will document at the cellular level the cumulative effect of AA (or AA isoforms) on membrane excitability and viability. If one or more of the isoforms modulates channel activity, action potential firing and/or cell survival, we will have identified a new signaling molecule(s) that may serve as a novel therapeutic target for treating ischemia. Inappropriate electrical activity gives rise to devastating brain disorders including, epilepsies and cell death following ischemia. These diseases trigger damaging inflammatory responses that elevate free arachidonic acid (AA) levels. We have discovered what appear to be novel isoforms of AA in a peripheral ganglion that regulates blood flow to the brain. By understanding the roles that AA and the novel AA isoforms serve in modulating nerve cell excitability, growth and survival, we hope to identify therapeutic targets that will minimize nerve cell death due to inflammatory signaling. [unreadable] [unreadable] [unreadable]