Inhibitory synaptic transmission in nociceptive neurons is largely mediated by glycine receptors (GlyRs). It has been shown recently that intracellular Ca2+ causes a large potentiation GlyR-mediated responses. There is good evidence suggesting that the potentiation is mediated by a diffusible cytoplasmic protein (CytoP). The goal of this project is to develop a new technology to identify the CytoP and study its interactions with GlyRs in trigeminal neurons. We hypothesize that a diffusible CytoP binds to GlyRs at low intracellular concentrations ([Ca2+]i), keeping GlyRs in a low activity state. When [Ca2+]i rises, the protein is rapidly dissociated from GlyRs, resulting in an enhancement of channel activity. To test this hypothesis, we will combine molecular biology, patch-clamp and imaging techniques to determine these interactions in molecular details. The CytoP will be isolated by the two-hybrid method. The interaction of the CytoP with GlyRs will be determine by simultaneously monitor (i) GlyR-mediated currents using the patch clamp technique, (2) intracellular Ca2+ transients using fluorescent Ca2+indicators and (iii) dynamic interactions between GlyRs and cytoplasmic proteins using the frequency resonance energy transfer (FRET) imaging technique. We will then extend this technology to identify CytoP and study the GlyR-CytoP interaction in trigeminal neurons. A better understanding of the mechanism of GlyR modulation will provide important information about the neuronal signaling and may lead to new therapy for orofacial pain.