The long range goal of the laboratory is to enhance our understanding of how neurotransmitters modulate calcium channel activity and the functional consequences of this modulation. The main inhibitory neurotransmitter in the mammalian central nervous system, ?-aminobutyric acid GABA) is known to attenuate N-type calcium currents by activation of GABAB receptors. A unique finding in our laboratory is that L-type current is facilitated in response to GABAB receptor activation in the neonatal neurons of the rat hippocampus. Understanding the mechanisms and functional significance of this facilitation will have significant consequences for the treatment of diseases such as drug addiction, epilepsy, insomnia and anxiety that are routinely treated with compounds interacting with the GABAergic system. The central hypothesis to be tested that GABAB receptors facilitate calcium current by a direct protein kinase C (PKC) mediated phosphorylation of Cav 1.2 or 1.3.
Specific aim #1 is to continue our studies on the signal transduction mechanism by determining if GABAB receptor activation facilitates L-type current through a specific PKC isoform. This will be accomplished by testing various inhibitors of the signaling pathway using whole cell patch clamp recording and calcium imaging. The activation of PKC isoforms will be tested by observing translocation from the cytosol to the membrane and phosphorylation of the isoforms using Western blot analysis.
Specific aim #2 is to determine whether the enhancement of L-type calcium current is through activation of silent L-type calcium channels via a phosphorylation event. These experiments will utilize single channel recording as well as Western blot analysis to observe phosphorylation of the L-type calcium channels. The experiments outlined in this proposal will provide knowledge about the signal transduction mechanism underlying the facilitation of L-type current and will provide information on the specific mechanism by which the facilitation of whole cell current occurs. A better understanding of the GABA-B receptor function may allow more subtle manipulation of the GABAergic system allowing for better design of pharmaceuticals with fewer side effects.
The experiments outlined in this proposal will increase our understanding of how the inhibitory neurotransmitter GABA affects neuronal function by modulation of voltage dependent calcium channels, particularly in the early neonatal period. Pharmaceutical compounds that interact with GABA receptors are routinely prescribed for anxiety, epilepsy, insomnia, etc. but these compounds also cause side effects such as drowsiness, mental slowing, and double vision. By increasing our understanding of the specific effects of GABA, it will be possible to design better drugs for these types of disorders.
