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.
