Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance

Abstract

Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

Survival in a bad neighborhood: pancreatic islets in cystic fibrosis

“…The two CFTR inhibitors used in the above studies, CFTR(inh)-172 and GlyH-101, are not specific for CFTR activity at the concentrations employed, 10 μM (Guo et al 2014), and 10-40 and 40-50 μM respectively (Edlund et al 2014). Both compounds inhibit mitochondrial function at 10 μM (Kelly et al 2010) and the activity of other chloride channels at 5 μM (Kelly et al 2010, Melis et al 2014, Friard et al 2017and reviewed in Di Fulvio et al 2014). Furthermore, 20 μM CFTR(inh)-172 has been shown to reduce glucose-stimulated calcium currents and insulin secretion in CFTR-KO ferret islets (GlyH-101 not tested) (Sun et al 2017), indicating that this compound, at the concentration used, likely has islet actions which are independent of CFTR.…”

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties

“…CFTR was recently reported to be directly (in a manner independent of PKA) activated by membrane stretch (Zhang et al, 2010a) and osmotic cell swelling (Xie et al, 2016). The conclusions deduced in both studies were mainly based on pharmacological observations using CFTR inh -172 (3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone) and GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4dihydroxyphenyl)methylene)glycine hydrazide] as CFTRspecific antagonists, although these drugs were known to also block VSOR (Melis et al, 2014;Friard et al, 2017) and ClC-2 (Cuppoletti et al, 2014). Moreover, these reports did not provide enough biophysical evidence to convince the readers that recorded channel events exhibit the phenotypic characteristics of CFTR.…”

Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function

“…CaCCinh-A01 can completely block CaCC current in human bronchial and intestinal epithelial cells, whereas T16Ainh-A01 poorly inhibits total CaCC current, but blocks an initial agonist-stimulated transient chloride current [34]. CaCCs are activated by an increases in the level of intracellular free calcium, and Cl − flux through CaCCs is generally thought to be driven by ANO1 or ANO2 [8]. An important finding of this study was that T16Ainh-A01 inhibited Cl − influx into the nucleus, while CaCCinh-A01 reduced Cl − level in intracellular compartments around the nucleus.…”

“…CaCCinh-A01 and T16Ainh-A01 are the most potent inhibitors of calcium-activated Cl − conductance and are typically used to examine the contributions of CaCCs/ANO1 in physiological models, although their specificity remains uncertain [8,16]. CaCCinh-A01 can completely block CaCC current in human bronchial and intestinal epithelial cells, whereas T16Ainh-A01 poorly inhibits total CaCC current, but blocks an initial agonist-stimulated transient chloride current [34].…”

“…The fibroblast properties of migration, secretion, and proliferation can be regulated by the activation of ion channels [5]. Meanwhile, chloride channels play essential roles in a variety of physiological functions including epithelial secretion, smooth muscle contraction, and sensory transduction [8]. Increasing evidence indicates that calcium-activated chloride channels (CaCCs) may participate in CF function [9][10][11] and be potential drug targets for the treatment of cardiovascular diseases [12].…”