We have been studying the effects of endocannabinoids such as anandamide and 2-AG on ligand-gated ion channels, potassium channels, and G-protein couple receptors. Our work has focused on three receptor coupled ligand-gated channels, the alpha-7 and alpha-4-beta-2 subtypes of nicotinic ACh receptors, 5-HT3 receptors and GABA receptors. In other studies we have examined ATP-sensitive potassium (KATP) channel. The KATP channels form an important link between metabolic state and cell excitability. They are implicated in the control of insulin secretion, vasoconstriction, cardiac rhythmicity, and oocyte maturation. We have found that anandamide inhibits the function of KATP channels in Xenopus oocytes and suppress the maturation process. Similar results were obtained in mammalian cell lines expressing KATP channels. KATP channels mediate glucose induced insulin release. In on-going collaborative studies, we are studying the effects of endocannabinoids on these channels and insulin secretion processes. [unreadable] [unreadable] In our earlier studies, using the Xenopus oocyte expression system, we were the first to show that alpha-7 nicotinic ACh, 5-HT3, and Glycine receptors are directly modulated by endocannabinoids. Recently, we have directed our research to mammalian cell lines and synaptosomes from the thalamic region of mice brain. Our work has shown that the endocannabinoid, anandamide, potently inhibits the function of the 4-beta-2 nACh receptor subtype expressed in SH-EP1 cells. Furthermore, similar effects of anandamide were observed on Rb efflux mediated by the activation of alpha-4-beta-2 nACh receptors in thalamic synaptosomes. Currently, we are extending our studies to investigate the role of direct cannabinoid modulation of alpha-7 and alpha-4-beta-2 nicotinic ACh receptors, 5-HT3 receptors and various GABA receptor subtypes on synaptic transmission and circuit behaviours of brain slices. [unreadable] [unreadable] We have initiated studies examining endocannabinoid modulation of the dopamine transporter (DAT); a membrane bound protein that regulates extracellular concentrations of dopamine in the CNS. Earlier studies indicated that the effects of cannabinoids on the function of DAT are not altered by cannabinoid receptor antagonists. Using a novel live cell imaging technique that enables visualization of DAT function in real-time, we have obtained evidence that anandamide inhibits DAT function. This effect is pertussis txoin independent indicating that it occurs independently of Gi/Go coupled receptors. Data regarding the influence of anandamide on trafficking of the transporter between the membrane and cytosol suggest that this effect is mediated, at least in part, via an increase in transporter internalization. [unreadable] [unreadable] Collaborative studies with Drs. Sanders-Bush and Zhang are examining endocannabionoid modulation of serotonin type 2C and mu-opioid receptor coupling to GIRK channels. We have found that anandamide inhibits the coupling of these channels to GIRK channels and other effectors. Since the activation of serotonin type 2C induces endocannabinoid synthesis, it is likely that anandamide inhibition of receptor-effector coupling constitutes a novel inhibitory feedback mechanism for these G-protein coupled receptors.[unreadable] [unreadable] The antinociceptive effects of cannabinoids are well established. However, in many pain models tested, the effects of cannabinoids are not reversed by cannabinoid receptor antagonists. Interestingly, receptors (e.g., glycine and GABA) and several Ca2+ channel subtypes that are implicated in nociceptive processing are potently modulated by anandamide and THC in a cannabinoid-receptor independent manner. Thus, we are investigating the role of cannabinoid modulation of these ion channels in cannabinoid-induced antinociception. We have developed a nociceptive dorsal root ganglion sensory neuronal line that enables us to examine endocannabinoid modulation of these ion channel functions in processing of nociceptive stimuli. Currently, we are testing the effects of endocannabinoids on the functional properties of ion channels expressed in these cell lines.