Biophysics and Structure-Function Relationships of LRRC8-Formed Volume-Regulated Anion Channels

Abstract

Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B–E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.

A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength

“…Here we present an intra-complex FRET sensor to monitor VRAC activity in live cells with subcellular resolution. The relationship of the observed FRET changes to VRAC gating are clear from numerous common features (Akita and Okada, 2014; König and Stauber, 2019; Pedersen et al, 2016; Strange et al, 2019): (i) the reversible decrease in FRET matches reversible VRAC activation upon treatment with hypo-osmotic solutions, and with the same kinetics as simultaneously-measured whole-cell VRAC currents, (ii) the intensity of the FRET decrease correlates with the applied extracellular hypotonicity; (iii) the modulation of VRAC currents, for example by cholesterol depletion, is mirrored by the observed FRET alteration; (iv) after activation with PMA, FRET rises in hypertonicity as VRAC is inactivated by cell shrinkage. FRET sensors unrelated to VRAC are not affected by hypo-osmotic solutions, demonstrating that the observed changes with our FRET sensor are indeed specific for VRAC activation.…”

“…When cells were treated with the DAG kinase inhibitor DOG, VRAC remained active in isotonic buffer after activation by hypotonicity, even though Γ i recovered to normal levels. The notion that ionic strength directly activates VRACs (Sabirov et al, 2000; Voets et al, 1999) has been controversial (König and Stauber, 2019; Strange et al, 2019). Several studies reported VRAC activation when isotonic fluid was injected into, or inactivation when fluid was withdrawn from the cells without altering the intracellular ionic strength (Best and Brown, 2009; Cannon et al, 1998; Poletto Chaves and Varanda, 2008; Zhang and Lieberman, 1996).…”

“…Prior to cloning, the biophysical properties of VRAC-mediated chloride currents (I Cl,swell ) and osmolyte conductance were studied extensively using mainly electrophysiological and pharmacological methods (König and Stauber, 2019; Pedersen et al, 2016; Strange et al, 2019); but their activation mechanism remained enigmatic for decades, in the absence of an unequivocal molecular identity for the channel. Numerous studies proposed contributions from membrane tension, the actin cytoskeleton and signaling pathways involving reactive oxygen species, calcium, G-proteins and phosphorylation cascades (Akita and Okada, 2014; Chen et al, 2019; Pedersen et al, 2016).…”

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

“…Volume regulated anion channel (VRAC) is a ubiquitously expressed chloride channel that has attracted much attention since the molecular structure of VRAC has been identified [4][5][6]. A growing body of evidence indicate that VRAC and their obligatory subunit, LRRC8A have critical roles in many cell functions including cell motility, proliferation, apoptosis, drug and metabolite transport, angiogenesis, and spermatid development, as well as in cell pathophysiological cell functions such as cancer drug resistance, ischemic brain edema, and glaucoma [7][8][9][10][11][12][13][14][15][16][17]. While the molecular structure of VRAC is well documented [18], understanding how VRAC expression at the membrane is regulated has been poorly explored due to lack of appropriate methodology.…”

Tweety Homologs (TTYH) Freshly Join the Journey of Molecular Identification of the VRAC/VSOR Channel Pore

“…In addition, LRRC8C, 8D or 8E was later found to be required together with LRRC8A for functional VSOR activity [51011]. The pore-forming roles of these LRRC8 members were suggested by discernible, though not drastic, alterations of ion selectivity by some point mutations and evidenced by successful reconstitution of anionic channel activity as well as by recent cryo-EM structure studies with purified LRRC8s, as summarized recently [1213]. However, it must be pointed out that several important properties (such as their cytoplasmic ATP independence, smaller single-channel conductance, and little voltage-dependent inactivation kinetics) of LRRC8-reconstituted channels are distinct from those of native VSOR channel, as summarized recently [914].…”