The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

Abstract

The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl-) current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A); the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their “swell activated” Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

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

“…However, this channel activity is somewhat different from the native VSOR activity, in light of its small single-channel conductance (approximately 8 pS), independence of intracellular ATP, and lack of inactivation kinetics. Such nonspecific activating effects of reduced ionic strength were also found on non-VSORtype anionic currents residually observed in neutrophils isolated from ebo/ebo mice in which LRRC8A truncation mutants naturally occur (Behe et al, 2017).…”

“…Second, in the cell-attached configuration, membrane stretch induced by applying negative pressure via a patch pipette failed to activate VSOR on nonswollen Intestine 407 cells and did not affect single-channel activity on the swollen cells (Okada, 1997). A lack of association between stretch-activated and swellingactivated Cl 2 currents was also demonstrated in human hepatocellular carcinoma cells (Mao et al, 2011) and in human neutrophils (Behe et al, 2017). (Sabirov et al, 2001;Kurbannazarova et al, 2011).…”

“…Subsequently, knockdown of mouse LRRC8A was also demonstrated to greatly reduce VSOR activity in a mouse C127 cell line and in primary cultured mouse nodose ganglia neurons (Wang et al, 2017b). More recently, mouse VSOR activity was found to be drastically diminished in T cells from Lrrc8a 2/2 mice and ebo/ebo mice with a Lrrc8a frameshift mutation and in neutrophils from ebo/ebo mice (Behe et al, 2017). Thus, it is evident that LRRC8A is an essential core component of VSOR not only in human cells but also in rodent cells.…”

“…IAA-94, indanyloxyacetic acid 94. polyunsaturated fatty acid, arachidonic acid (IC 50 = 8 mM; Kubo and Okada, 1992); oxonol dyes, bis-(1,3dibutylbarbituric acid)pentamethine oxanol (diBA-(5)-C 4 ) (IC 50 = 1.8 mM; Arreola et al, 1995) and WW781 [4-[4-[(1E,3E)-5-(1,3-dibutyl-2,4,6-trioxo-1,3-diazinan-5-ylidene)penta-1,3-dienyl]-3-methyl-5-oxo-4H-pyrazol-1yl]benzenesulfonic acid] (IC 50 = 25 mM; Behe et al, 2017); an antimalaria drug, mefloquine (IC 50 = 1.2 mM; Maertens et al, 2000); a hemichannel blocker, carbenoxolone (CBX) (IC 50 = 15.4 mM; Benfenati et al, 2009); and cell-permeable CFTR inhibitors, GlyH-101 (IC 50 = 5 to 6 mM; Melis et al, 2014) (IC 50 = 9.5 mM; Friard et al, 2017) and 59: 3,4]pyrrolo[1,2-a]quinoxaline-8,10(5H,9)-dione] (IC 50 = 19.6 mM; Friard et al, 2017). Inhibition of VSOR was also reported to be induced by a purinergic P2X receptor blocker, pyridoxalphosphate-6-azophenyl-29,49-disulfonic acid (Galietta et al, 1997;Darby et al, 2003;Poletto Chaves and Varanda, 2008), in a voltage-dependent manner and by unprotonated quinidine in a voltageindependent manner (at pH 9; Voets et al, 1996;Behe et al, 2017). Since the drugs mentioned in this paragraph have no obvious common structural motives, we assume that VSOR channel protein has several pockets (binding sites) of different shapes and electrostatics to accommodate chemical substances of very different structures.…”

Subunit‐dependent oxidative stress sensitivity of LRRC8 volume‐regulated anion channels

“…Also neutrophils from ebo mice retained phagocytic activity and had normal‐sized vacuoles (Behe et al . ).…”