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Canonical transient receptor potential 4 and 5 (TRPC 4 and TRPC5) are non-selective cation channels widely expressed in many mammalian tissues.Activation of these channels causes membrane depolarization and intracellular Ca2+ increases.TRPC4 has been implicated in neurotransmitter release from thalamic interneurons, endothelial-dependent regulation of vascular tone, microvascular permeability, and excitation-contraction coupling of intestinal smooth muscles while TRPC5 has been shown to be important for neurite extension and growth cone morphology of hippocampal neurons and behavior responses to fear conditioning.Both channels are thought to be responsible for afterdepolarization of cerebral cortex and hippocampal neurons.Using whole-cell patch clamp recording and fluorescence membrane potential measurements, we show that channel activation of TRPC4 and C5 are dependent on the co-stimulation of Gi/o-and Gq/11-coupled receptors.The effect of Gi/o proteins appears to be mainly mediated through the activated form of Gαi/o rather than Gβγ.Taking the advantage of Gi/o-induced TRPC4 activation and the robust Ca2+ influx signal, we have developed a cell-based assay for high throughput screening of TRPC channel ligands.Primary screening was performed at the Johns Hopkins Ion Channel Center against the Molecular Libraries Small Molecule Repository (MLSMR), yielding >1000 lead compounds as potential agonists, antagonists and allosteric modulators.We have performed detailed characterizations on two antagonists, showing excellent selectivity to TRPC4/C5 as compared to other TRP channels and voltage-gated K+, Na+, and Ca2+ channels.These compounds will be valuable tools for dissecting the mechanisms of regulation and physiological functions of these novel channels.