Glycine receptors (GlyRs), together with GABAA and nicotinic ACh receptors, form part of the ligand-activated ion channel superfamily and play an important role in the excitability of the mammalian brain stem and spinal cord. For instance, the activation of glycine receptors (GlyRs) in brain stem, spinal cord and brain provides a main inhibitory control for neuronal excitability. Studies from our and other laboratories have shown that clinically relevant concentrations of ethanol (1-1 O0 mM) enhanced the function of these receptors in hippocampal, cortical and spinal neurons. We have recently published that GlyRs are regulated by G protein activation. In addition, our study indicated that this novel modulation of GlyR was via the G-beta-gamma dimer. The mechanisms by which ethanol is able to affect GlyRs had remained largely unknown. An important line of research has dealt with the search of sites that permit the binding of ethanol within the GlyR. For instance, single residue mutations in S267 within the alpha1 GlyR was reported to change the sensitivity to 200 mM ethanol by more than 50%. Overall, these studies concluded that ethanol effects on GlyRs could be related to the amino acid sequence inTM2 andTM3: Based on our recent paper and preliminary results, we have proposed the hypothesis that the effects of low ethanol concentrations depend on the activation of G proteins, more than the presence of sites that directly bind the ethanol molecule, For example, GlyR sensitivity to ethanol was significantly modified by changing the stoichiometry of the heterotrimeric complex: More importantly, the potentiation of the GlyR was completely blocked by overexpression of Gl3y subunits (occlusion), as well as a G-beta-gamma scavenger peptide (ct-GRK). In order to characterize the importance- of G protein activation on ethanol sensitivity; we will study G protein modulators using heterologous expression of G protein subunits in HEK cells, electrophysiology, receptor mutagenesis and Western blot techniques. Key experiments will be performed in cultured spinal and dorsal root ganglion (DRG) neurons to confirm the major effects in a neuronal substrate. Such information will help us to understand the mechanisms by which ethanol affects these inhibitory receptors, which are important in functions such as convulsions, sensory integration, muscle tone and respiration. In summary, this proposal may provide support for a novel mechanism of ethanol effects in motor and sensory functions.
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