The GABA-A receptor/ionophore complex is responsible for most fast inhibition in the mammalian brain. Activation of this receptor increases the flux of chloride across the neuronal plasma membrane, which typically hyperpolarizes the neuron and diminishes the probability of firing action potentials. Several modulatory sites that modify the effect of GABA on this receptor/ionophore complex have been identified over the last decade. Drugs that act at these sites and diminish GABA-induced currents are usually convulsants; conversely, drugs that potentiate GABA currents are often anticonvulsants. We are particularly interested in a picrotoxin site(s) and its modulation by a series of gamma-butyrolactones that have been synthesized in our laboratories. We have previously shown that different substitute butyrolactones can either enhance (""""""""GABA+"""""""") or block (""""""""GABA-"""""""") GABA-induced chloride currents. In the electrophysiology core of this program project application, we plan to: 1. Characterize new gamma-butyrolactones that appear to have promise as clinical anticonvulsants. We are particularly interested in studying derivatives of the compound aflatrem, which is almost 1000 times more potent than other gamma-butyrolactones in augmenting GABA-induced currents. We shall also investigate lactone optical isomers, which may be more potent and selective for this site. 2. Define the detailed mechanism(s) of action of these compounds. We believe these compounds act at two distinct sites that are within the M2 transmembrane segment of the GABA-A subunits. We shall use a variety of GABA-A receptor subunit mutants to attempt to confirm this. We shall also investigate the hypothesis that arachidonic acid may be a natural ligand for this site. 3. Evaluate the modulation of other ion channels by this class of compounds. We already know that certain lactones potentiate nicotinic cholinergic currents in hippocampal neurons. Based on sequence homology with the GABA-A receptor subunits, we expect that glycine, serotonin, and homomeric receptors composed of pi1 GABA subunits should also respond to gamma-butyrolactones. The planned experiments will use standard electrophysiological techniques to characterize GABA-A receptors and inhibitory currents in rat neurons and also receptor subunits transfected into a kidney cell line. We expect this work to have therapeutic implications for a number of medical, neurological, and psychiatric diseases, especially epilepsy.
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