The ultimate goal of the proposed research project is to fully characterize the ion channels that allow chloride ions to pass into and out of rat and human cerebral cortical neurons. Most electrophysiological research has focused on ion channels involved in the transport of cations such as sodium, potassium, and calcium. It is now clear that anion transport is at least as important to the normal and pathological functions of cells as is the transport of cations and therefor much research is needed to determine the basic mechanisms of the proteins that allow Cl and other anions to pass into and out of cells. Excitable cells, such as neurons and muscle, are especially sensitive to anion fluxes as the movements of anions affects the excitability of these cells and contributes to the integration of excitatory and inhibitory signals impinging on these cells. The predominant non-transmitter activated anion-selective ion channel present in neurons is the so-called """"""""fast"""""""" Cl channel, previously described in tissue-cultured rat skeletal muscle, and recently discovered by this laboratory to be present at high density in rat and human cerebral cortical neurons. In a previous study, the kinetic activity of these channels was determined and the voltage dependence was analyzed.
The specific aims of the current proposal are to (1) expand the discovery that these Cl channels are blocked, in a voltage-dependent manner by the classical K channel blocker, tetraethylammonium ion (TEA) and other quaternary ammonium (QA) ions (2) study kinetic activity of Cl channels as low temperatures that facilitate the resolution of the very rapid conformational rearrangements that the ion channel proteins undergo during the ion transport process; (3) determine the physiological role of fast Cl channels in the functioning of neurons, and to confirm and expand upon the discovery that these channels contribute to neuronal volume regulation and (4) to attempt to clone the neuronal fast Cl channel using an oocyte expression assay. The research proposed will significantly increase our knowledge about anion transport channels in the nervous system and may lead to a better understanding about how these channels are involved in the regulation of neural functions in normal and pathological states.
