The long term objective of this research is a better understanding of the mechanisms of action of antiepileptic drugs used in the treatment of epilepsy. The proposed studies will concentrate within the fields of neurophysiology, neuropharmacology and molecular biology. Experiments will be designed to study the basic action of antiepileptic drugs on voltage dependent and transmitter gated ion channels. Voltage dependent calcium channels will be studied in mouse and rat primary and acutely dissociated cell cultures of central and primary afferent neurons. The calcium current components recorded in these tissues include a transient low threshold current (T), a large transient high threshold current (N), and a slowly inactivating high threshold current (L). Using the whole cell voltage clamp technique, we will determine the effect of all clinically used antiepileptic drugs, active metabolites, and pentylenetetrazol on T, N, and L whole cell calcium currents. Linear leak and capacitance currents will be subtracted from recorded currents followed by analysis of peak currents, activation and inactivation kinetics, voltage dependency, and the time course of recovery from inactivation. Using the cell attached single channel recording technique, we will determine the properties of voltage dependent T, N, and L calcium channels and the effects of antiepileptic drugs, active metabolites, and pentylenetetrazol on single channel currents. Recorded data will be digitized and analyzed off line by computer. Individual currents will be analyzed by the distribution of channel amplitudes, open and closed time histograms and burst characteristics including frequency, duration, distribution, number and types of openings and closings, and interburst interval. Transmitter gated chloride channels will be studied in Xenopus oocytes by intracellular recording techniques after injection and expression of either isolated or cloned GABA A receptor mRNA. Single channel currents will be studied in the oocytes and in acutely and stably cDNA transfected mammalian cell lines. Emphasis will be placed on the characterization of expressed receptors' responses to benzodiazepines, barbiturates, and convulsant drugs and on the analysis of kinetic properties.
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