GABAA-mediated synaptic inhibition plays an important role in many normal physiological processes in the CNS and appears to be critically important in the physiology of epilepsy. The development of epileptic activity, the onset of seizures in an epileptogenic area, and the spread of seizures to normal brain are all either due to, or are associated with, a decrease in inhibitory efficacy. The factors which regulae GABAA mediated inhibition at the level of the postsynaptic receptor are only partially understood and the C1 channels which underlie the GABA response are only partially characterized. Our previous work has been devoted to characterizing GABAA-mediated inhibition in cultured neocortical and hippocampal neurons, at the cellular and molecular level, using intracellular, whole cell patch clamp and single channel recording techniques. We will continue these studies, focusing initially on the molecular mechanisms responsible for desensitization and resensitization of the GABAA receptor at both the cellular and single channel level. We will determine if these processes are related to changes in Cai or cAMP, activation or inhibition of protein kinases or phosphatases, or modulation of G proteins or phosphoinositol pathways. We will describe the properties of the GABA-activated C1 channel, including the size of the main conducting state, the presence of other substates, the kinetics of channel openings and closing, including tendency to open in bursts, and other physiological influences on the channel. We will also determine if desensitization is due to a change in single channel conductance, a change in probability of openings or in burst versus single opening, a change in open times or inactivation of one subtype of C1 channel (as we have recently demonstrated for desensitization at the quisqualate receptor.) The mechanisms of action of several important drugs which work via the GABA receptor complex, including the benzodiazepines, barbiturates, beta- carbolines, and progesterone metabolites will be analyzed at the channel level and we will determine if neuropeptides which co-exist with GABA in cortical neurons have direct modulatory effects on the GABA receptor and C1 channel. We will also determine whether activation of glutamate receptor subtypes affects GABA responses and whether GABA activation influences the changes in CAi induced by excitatory neurotransmitter (NT) action. Finally, if GABA responses are affected by second messenger pathways, we will determine how these effects may be mediated by other NTs which affect these systems, especially NE, DA, SOM, VIP and CCK. It is hoped that by understanding the regulation of GABAA-mediated inhibition at the molecular level we will be able to devise new strategies for preventing the loss of inhibition which appears to promote the development and spread of seizures.
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