title: Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.
abstract: Transient receptor potential canonical (TRPC) channels are Ca(2+)-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 was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), 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 μm 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 GTPγS, 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.
authors.affiliation: Department of Neuroscience, High Throughput Biology Center, and Johns Hopkins Ion Channel Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Miller
authors.forename: Jie
authors.initials: J
authors.lastname: Shi
authors.forename: Yingmin
authors.initials: Y
authors.lastname: Zhu
authors.forename: Maksym
authors.initials: M
authors.lastname: Kustov
authors.forename: Jin-bin
authors.initials: JB
authors.lastname: Tian
authors.forename: Amy
authors.initials: A
authors.lastname: Stevens
authors.forename: Meng
authors.initials: M
authors.lastname: Wu
authors.forename: Jia
authors.initials: J
authors.lastname: Xu
authors.forename: Shunyou
authors.initials: S
authors.lastname: Long
authors.forename: Pu
authors.initials: P
authors.lastname: Yang
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Zholos
authors.forename: James M
authors.initials: JM
authors.lastname: Salovich
authors.forename: C David
authors.initials: CD
authors.lastname: Weaver
authors.forename: Corey R
authors.initials: CR
authors.lastname: Hopkins
authors.forename: Craig W
authors.initials: CW
authors.lastname: Lindsley
authors.forename: Owen
authors.initials: O
authors.lastname: McManus
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
mesh_terms: D000818:Animals; D002412:Cations / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D007422:Intestines / Q000166:cytology; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D002412:Cations; D005456:Fluorescent Dyes; D007211:Indoles; C567966:ML 204; D010880:Piperidines; D011976:Receptors, Muscarinic; D017450:Receptors, Opioid, mu; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C112021:Trpc5 protein, mouse