DCPIB, an Inhibitor of Volume-Regulated Anion Channels, Distinctly Modulates K2P Channels

Lv, Jin-Yan; Liang, Yemei; Zhang, Shiqing; Lan, Qunsheng; Xu, Zhiwu; Wu, Xiaoyan; Kang, Lijun; Ren, Jianmin; Cao, Ying; Wu, Ting; Lin, Kaili; Yung, Kin Lam; Cao, Xiong; Pang, Jianxin; Zhou, Pingzheng

doi:10.1021/acschemneuro.9b00010

Cited by 29 publications

(25 citation statements)

References 34 publications

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“…As with any such another key point raised by our findings is that the current array of VRAC inhibitors are not sufficiently selective for this purpose. Indeed, we and others have demonstrated extensive undesirable off-target activity in even the current gold-standard inhibitor, DCPIB (Afzal et al, 2019;Lv et al, 2019;Minieri et al, 2013). As such, the discovery of compounds with greater selectivity for VRAC over other chloride channels would be of great benefit, both to better understand VRAC's roles in physiology and to explore its value as a therapeutic target.…”

Section: Discussionmentioning

confidence: 99%

LRRC8Ais dispensable for a variety of microglial functions and response to acute stroke

Cook

Gray

Lemarchand

et al. 2022

Glia

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Microglia, resident brain immune cells, are critical in orchestrating responses to central nervous system (CNS) injury. Many microglial functions, such as phagocytosis, motility and chemotaxis, are suggested to rely on chloride channels, including the volume‐regulated anion channel (VRAC), but studies to date have relied on the use of pharmacological tools with limited specificity. VRAC has also been proposed as a drug target for acute CNS injury, and its role in microglial function is of considerable interest for developing CNS therapeutics. This study aimed to definitively confirm the contribution of VRAC in microglia function by using conditional LRRC8A‐knockout mice, which lacked the essential VRAC subunit LRRC8A in microglia. We demonstrated that while VRAC contributed to cell volume regulation, it had no effect on phagocytic activity, cell migration or P2YR12‐dependent chemotaxis. Moreover, loss of microglial VRAC did not affect microglial morphology or the extent of ischemic damage following stroke. We conclude that VRAC does not critically regulate microglial responses to brain injury and could be targetable in other CNS cell types (e.g., astrocytes) without impeding microglial function. Our results also demonstrate a role for VRAC in cell volume regulation but show that VRAC is not involved in several major cellular functions that it was previously thought to regulate, and point to other, alternative mechanisms of chloride transport in innate immunity.

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Section: Discussionmentioning

confidence: 99%

LRRC8Ais dispensable for a variety of microglial functions and response to acute stroke

Cook

Gray

Lemarchand

et al. 2022

Glia

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“…Several studies reported an impairment of proliferation and/or migration of various cell lines in the presence of VRAC inhibitors [24,25,28,29,30,31,32,33,34,35,36,37,38]. However, the available VRAC inhibitors display little selectivity and often also inhibit other anion channels [55,56], or as in the case of the potent and relatively selective VRAC blocker DCPIB [57] even modulate potassium channels [58,59]. The identification of LRRC8 proteins as essential VRAC components [4,5] enabled investigating physiological functions of VRAC by molecular biological tools.…”

Section: Discussionmentioning

confidence: 99%

The Volume-Regulated Anion Channel LRRC8/VRAC Is Dispensable for Cell Proliferation and Migration

Liu

Stauber

2019

IJMS

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Cells possess the capability to adjust their volume for various physiological processes, presumably including cell proliferation and migration. The volume-regulated anion channel (VRAC), formed by LRRC8 heteromers, is critically involved in regulatory volume decrease of vertebrate cells. The VRAC has also been proposed to play a role in cell cycle progression and cellular motility. Indeed, recent reports corroborated this notion, with potentially important implications for the VRAC in cancer progression. In the present study, we examined the role of VRAC during cell proliferation and migration in several cell types, including C2C12 myoblasts, human colon cancer HCT116 cells, and U251 and U87 glioblastoma cells. Surprisingly, neither pharmacological inhibition of VRAC with 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB), carbenoxolone or 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), nor siRNA-mediated knockdown or gene knockout of the essential VRAC subunit LRRC8A affected cell growth and motility in any of the investigated cell lines. Additionally, we found no effect of the VRAC inhibition using siRNA treatment or DCPIB on PI3K/Akt signaling in glioblastoma cells. In summary, our work suggests that VRAC is dispensable for cell proliferation or migration.

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“…Unfortunately, DCPIB is unable to cross the blood–brain barrier when administered intravenously and suffers from poor selectivity (Zhang et al, ) DCPIB inhibits VRAC activity with an IC 50 of approximately 5 µM, making it the most potent and best‐in‐class VRAC inhibitor currently available. However, at concentrations used to inhibit VRAC, DCPIB also inhibits H‐K‐ATPase (Fujii et al, ), inward rectifier potassium (Kir) channels (Deng, Mahajan, Baumgarten, & Logothetis, ), two pore‐domain potassium (K2P) channels (Lv et al, ), glutamate release via connexin hemichannels (Bowens, Dohare, Kuo, & Mongin, ), and glutamate uptake by GLT‐1 glutamate transporters (Bowens et al, ). Indeed, the molecular pharmacology of VRAC is plagued by weak and non‐selective inhibitors.…”

Section: Introductionmentioning

confidence: 99%

The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel

et al. 2019

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There has been a resurgence of interest in the volume-regulated anion channel (VRAC) since the recent cloning of the LRRC8A-E gene family that encodes VRAC.The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best-inclass VRAC inhibitor, DCPIB, suffers from off-target activity toward several different channels and transporters. Considering that some anion channel inhibitors also suppress mitochondrial respiration, we systematically explored whether DCPIB inhibits respiration in wild type (WT) and LRRC8A-knockout HAP-1 and HEK-293 cells. Knockout of LRRC8A had no apparent effects on cell morphology, proliferation rate, mitochondrial content, or expression of several mitochondrial genes in HAP-1 cells. Addition of 10 µM DCPIB, a concentration typically used to inhibit VRAC, suppressed basal and ATP-linked respiration in part through uncoupling the inner mitochondrial membrane (IMM) proton gradient and membrane potential.Additionally, DCPIB inhibits the activity of complex I, II, and III of the electron transport chain (ETC). Surprisingly, the effects of DCPIB on mitochondrial function are also observed in HAP-1 and HEK-293 cells which lack LRRC8A expression.Finally, we demonstrate that DCPIB activates ATP-inhibitable potassium channels comprised of heterologously expressed Kir6.2 and SUR1 subunits. These data indicate that DCPIB suppresses mitochondrial respiration and ATP production by dissipating the mitochondrial membrane potential and inhibiting complexes I-III of the ETC. They further justify the need for the development of sharper pharmacological tools for evaluating the integrative physiology and therapeutic potential of VRAC in human diseases. K E Y W O R D SDCPIB, LRRC8, mitochondria, respiration

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DCPIB, an Inhibitor of Volume-Regulated Anion Channels, Distinctly Modulates K2P Channels

Cited by 29 publications

References 34 publications

LRRC8Ais dispensable for a variety of microglial functions and response to acute stroke

LRRC8Ais dispensable for a variety of microglial functions and response to acute stroke

The Volume-Regulated Anion Channel LRRC8/VRAC Is Dispensable for Cell Proliferation and Migration

The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel

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