Burgess N. Christensen - US grants

Neurotransmitters can effect cell function by binding to receptors associated with ion channels (ionotropic receptors) or to receptors that stimulate second messenger production (metabotropic receptors). The gating of ion channels results in changes in membrane potential that alters cell excitability. Second messengers often bring about an increase in intracellular Ca2+. Cytoplasmic Ca2+ transients trigger key cellular events including modification of excitability, cell growth and transmitter release. The long-term objective of the proposed research is to understand the mechanisms of action of neurotransmitters on ion permeation and Ca2+ metabolism in fish retinal horizontal cells. The specific aims are to determine the basic mechanisms of ion permeation initiated through ligand gated channels, the functional components of the proteins that constitute these channels, the changes in intracellular Ca2+ initiated by neurotransmitters that may be important in regulating the electrical activity of horizontal cells and the neurotoxic response. Two approaches will be used. One employs whole cell voltage clamp using patch electrodes to measure the membrane current produced by applied neurotransmitters and pharmacological agents that modify either the action of the transmitters or the amino acid composition of the receptor/channel protein. The second method will utilize the Ca2+ sensitive fluorescent indicator fura-2 to measure intracellular Ca2+ concentration in the presence of neurotransmitters. These experiments will be done on voltage clamped cells so as to distinguish the contributions to the changes in intracellular Ca2+ made by voltage sensitive Ca2+ channels and ligand gated channels. To carry out these studies, a concentration clamp system has been developed that permits the rapid exchange of solutions while maintaining the cell under voltage clamp with simultaneous measurement of cell fluorescence. Under these conditions, precise control of known concentrations of drugs is maintained. The measure of cell viability as a function of [Ca2+]i will be determined by cell shape and cell response to agonists or changes in membrane voltage. The results from this research will provide new and extensive information on many of the structural and biophysical properties of neurotransmitter- receptor interactions. The results of this investigation will have health- related implications since neurons rely on interactions with chemical reagents for several key functions.

DESCRIPTION (Investigator's Abstract): Cytoplasmic Ca2+ transients trigger key cellular events including excitability, cell growth and transmitter release. The resting free Ca2+ concentration (Ca2+)1 is maintained by several buffer mechanisms that include Ca2+ binding proteins sequestration of Ca2+ into rapidly- and slowly-exchanging membrane-bound storage organelles (Ca2+ stores), membrane channels and Ca2+ pumps. The rapidly-exchanging Ca2+ stores of the chicken cerebellum Purkinje cell have been tentatively identified as a subset of smooth-surfaced endoplasmic reticulum and equipped with several protein constituents such as Ca2+-pump, Ca2+ release channels (sensitive either to inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine) and calsequestrin and intraluminal Ca2+ binding protein. The long-term objective of the proposed research is to define the functional role of intracellular rapidly-exchanging Ca2+ stores in chicken cerebellar Purkinje cells. To this end, we propose a multifaceted approach that shall accomplish three major goals. 1) The biochemical purification and characterization of Ca2+ stores will involve measurements of Ca2+ fluxes, and enzymatic and immunological binding assays. 2) The functional characterization of the Ca2+ release channels associated with the purified organelles will involve measurement of Ca2+ fluxes by isotope and spectrophotometric methods and single channel analysis. Moreover, the structure-function relationship of these Ca release channels will be studied by means of anti-peptide antibodies for specific domains of the channel molecule. 3) The definition of the role that plasma membrane receptors activated by excitatory amino acids and associated with either ion channels or second messenger systems play in redistribution of Ca2+ from intracellular Ca2+ stores. Excitatory amino acids will be applied in a concentration clamp system on enzymatically isolated Purkinje cells maintained under a voltage clamp to determine their capacity to increase (Ca2+)1 by allowing permeation through a ligand-gated channel or by activation of a second messenger system that releases Ca2+ from one of the rapidly exchanging intracellular Ca2+ stores. The results from this research will provide new and extensive information on many of the biochemical and biophysical properties of rapidly exchanging Ca2+ stores. The results of this investigation will have health-related implications since neurons rely on redistribution of Ca2+ from intracellular stores for several key functions.

0119544
Motamedi
Recent advances in nanotechnology and bioengineering have opened the exciting prospect for promoting direct interaction of electronics, optics and biological systems. The primary obstacle to realizing this goal in the fields of neuroscience and medical devices such as prostheses is the inadequate understanding of the molecular processes involved in coupling between living neurons and nanomaterials, and how to optimize the coupling between man made materials and living systems. In this project, the intent is to investigate the dynamics and mechanisms of the live/lifeless matter interaction in a model system consisting of a thin film composed of nanoparticles and cultured nerve cells. The multi-disciplinary and multi- institutional "Linked Collaborative Proposals" bring together several research groups with broad expertise and research interest to conduct experimental and theoretical studies aimed at characterizing the interaction that occur at the interface of nanomaterials and neurons and optimizing the interface for effective photon-activation of neurons following photonic probing of the interface of nanomaterials that are attached to the cells. The partners at Oklahoma State University lead by Dr. Kotov will carry out the materials science aspect of this project, while the investigation at the University of Texas Medical Branch lead by Dr. Motamedi will concentrate on the bioengineering and electrophysiological components of this work.

Specifically, the objectives of the project are the following. (1) Preparation of biocompatible nanoparticle multilayers that can be attached to nerve cells. (2) Registration and characterization of the photoinduced nerve cell membrane currents and potentials following optical excitation of the interface as function of NP and biological structures. (3) Optimization of NP-cell coupling for different interface structure.