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      CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor

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          Abstract

          Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 ( LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate G q-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- G q-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC 50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

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          Author and article information

          Journal
          Am J Physiol Cell Physiol
          Am. J. Physiol., Cell Physiol
          ajpcell
          Am J Physiol Cell Physiol
          AJPCELL
          American Journal of Physiology - Cell Physiology
          American Physiological Society (Bethesda, MD )
          0363-6143
          1522-1563
          1 October 2019
          7 August 2019
          1 October 2020
          : 317
          : 4
          : C857-C866
          Affiliations
          1Department of Pharmacology, Vanderbilt University , Nashville, Tennessee
          2Department of Anesthesiology, Vanderbilt University Medical Center; Nashville, Tennessee
          3Novo Biosciences, Inc. , Bar Harbor, Maine
          Author notes
          Address for reprint requests and other correspondence: J. S. Denton, T4208 Medical Center North, 1161 21st Ave. South, Vanderbilt Univ. Medical Center, Nashville, TN 37232 (e-mail: jerod.s.denton@ 123456vumc.org ).
          Article
          PMC6850990 PMC6850990 6850990 C-00281-2019 C-00281-2019
          10.1152/ajpcell.00281.2019
          6850990
          31390227
          Copyright © 2019 the American Physiological Society
          Funding
          Funded by: HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 10.13039/100000062
          Award ID: 1F31DK120225-01
          Award ID: R01 DK51610
          Categories
          Research Article

          HTS, VRAC, LRRC8, CysLT1R

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