Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Abstract Transient receptor potential canonical (TRPC) channels are Ca2+-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent and selective TRPC4 channel inhibitor. A high-throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository (MLSMR) was performed for inhibitors that blocked intracellular Ca2+ rise in response to stimulation of mouse TRPC4beta by mu-opioid receptors. ML204 inhibited TRPC4beta-mediated intracellular Ca2+ rise with an IC50 value of 0.96 uM and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4beta currents activated through either mu-opioid receptor stimulation or intracellular dialysis of GTP(gamma)S suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 uM of ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTP(gamma)S, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.