To better understand the basic mechanisms and pharmacology of the most important inhibitory neurotransmitter in the CNS, this study will investigate postsynaptic GABA-A receptor function under more physiologically relevant conditions. Patch clamp recording and ultrafast ligand application techniques will be utilized to: 1) characterize the single channel basis of GABA-A receptor inhibitory postsynaptic current (IPSC) responses, and 2) identify the single channel mechanisms underlying pharmacologic modulation of GABA-mediated IPSCs. The GABA-A receptor is composed of a protein complex that forms a chloride selective ion channel, and contains binding sites for regulation by GABA and by clinically important benzodiazepines, barbiturates, steroids and convulsants. Binding of GABA to receptors in the postsynaptic membrane evokes a macroscopic IPSC composed of a stochastic response resulting from the opening of many individual ion channels. IPSC amplitude and/or time course can be pharmacologically altered to modulate neuronal inhibition. The single channel basis for GABA-mediated IPSCs and for IPSC modulation by drugs has not been extensively studied. Previous studies have indicated that GABA and allosteric modulators of the GABA receptor altered single channel gating (opening and closing) kinetic properties, rather than channel conductance. However, since receptor desensitization may alter gating characteristics of the ion channel, these studies have been limited by relatively slow speed and long duration of ligand exposure to the receptor. At the synapse, release and uptake mechanisms result in rapid and probably transient GABA exposure to the postsynaptic receptor. Therefore, mechanisms of neurotransmitter and drug action would be better studied under conditions more similar to that found at the synapse such as proposed in this study.
Aims of this study are: 1) to characterize the kinetic properties and to develop a model of the single channel correlate of GABA-mediated IPSCs, 2) to identify the single channel mechanisms underlying pharmacologic regulation of GABA-mediated IPSCs, and 3) to characterize the single channel properties of GABA receptor agonist dependent desensitization as it is related to modulation of IPSC responses. Studies will be performed using newly developed ultrafast ligand application techniques on excised outside-out patches obtained from rat cortical neurons in culture. Previously developed single channel analysis techniques will be extended and applied in the investigations. This study will establish a model to understand and predict, under a variety of conditions, GABA-mediated and pharmacologically modulated IPSC responses based upon the stochastic activity of single GABA receptor channels. The long-term goal of this project is to provide a better understanding of synaptic GABA receptor function and of clinically relevant GABA receptor pharmacology.
