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

Melissa Miller, Jie Shi, Yingmin Zhu, Maksym Kustov, Jin Bin Tian, Amy Stevens, Meng Wu, Jia Xu, Shunyou Long, Pu Yang, Alexander Zholos V, James M. Salovich, C. David Weaver, Corey R Hopkins, Craig W. Lindsley, Owen McManus, Min Li, Michael X. Zhu

Research output: Contribution to journalArticle

103 Citations (Scopus)

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)triphos-phate (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.

Original languageEnglish (US)
Pages (from-to)33436-33446
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number38
DOIs
StatePublished - Sep 23 2011

Fingerprint

Small Molecule Libraries
Transient Receptor Potential Channels
Opioid Receptors
Ion Channels
Cations
Fluorescent screens
Voltage-Gated Sodium Channels
Voltage-Gated Potassium Channels
Signal transduction
Dialysis
Guanosine
Potassium Channels
Clamping devices
Spinal Ganglia
Carbachol
Muscarinic Receptors
Calcium Channels
Baths
Synaptic Transmission
Muscle Cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels. / Miller, Melissa; Shi, Jie; Zhu, Yingmin; Kustov, Maksym; Tian, Jin Bin; Stevens, Amy; Wu, Meng; Xu, Jia; Long, Shunyou; Yang, Pu; Zholos V, Alexander; Salovich, James M.; Weaver, C. David; Hopkins, Corey R; Lindsley, Craig W.; McManus, Owen; Li, Min; Zhu, Michael X.

In: Journal of Biological Chemistry, Vol. 286, No. 38, 23.09.2011, p. 33436-33446.

Research output: Contribution to journalArticle

Miller, M, Shi, J, Zhu, Y, Kustov, M, Tian, JB, Stevens, A, Wu, M, Xu, J, Long, S, Yang, P, Zholos V, A, Salovich, JM, Weaver, CD, Hopkins, CR, Lindsley, CW, McManus, O, Li, M & Zhu, MX 2011, 'Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels', Journal of Biological Chemistry, vol. 286, no. 38, pp. 33436-33446. https://doi.org/10.1074/jbc.M111.274167
Miller, Melissa ; Shi, Jie ; Zhu, Yingmin ; Kustov, Maksym ; Tian, Jin Bin ; Stevens, Amy ; Wu, Meng ; Xu, Jia ; Long, Shunyou ; Yang, Pu ; Zholos V, Alexander ; Salovich, James M. ; Weaver, C. David ; Hopkins, Corey R ; Lindsley, Craig W. ; McManus, Owen ; Li, Min ; Zhu, Michael X. / Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 38. pp. 33436-33446.
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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)triphos-phate (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.",
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AU - Tian, Jin Bin

AU - Stevens, Amy

AU - Wu, Meng

AU - Xu, Jia

AU - Long, Shunyou

AU - Yang, Pu

AU - Zholos V, Alexander

AU - Salovich, James M.

AU - Weaver, C. David

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AU - Li, Min

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