Inactivation and Anion Selectivity of Volume-regulated Anion Channels (VRACs) Depend on C-terminal Residues of the First Extracellular Loop

Abstract

Canonical volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have many other important roles, including tumor drug resistance and release of neurotransmitters. Although VRAC-mediated swelling-activated chloride currents (I Cl,vol ) have been studied for decades, exploration of the structure-function relationship of VRAC has become possible only after the recent discovery that VRACs are formed by differently composed heteromers of LRRC8 proteins. Inactivation of I Cl,vol at positive potentials, a typical hallmark of VRACs, strongly varies between native cell types. Exploiting the large differences in inactivation between different LRRC8 heteromers, we now used chimeras assembled from isoforms LRRC8C and LRRC8E to uncover a highly conserved extracellular region preceding the second LRRC8 transmembrane domain as a major determinant of I Cl,vol inactivation. Point mutations identified two amino acids (Lys-98 and Asp-100 in LRRC8A and equivalent residues in LRRC8C and -E), which upon charge reversal strongly altered the kinetics and voltage dependence of inactivation. Importantly, charge reversal at the first position also reduced the iodide > chloride permeability of I Cl,vol . This change in selectivity was stronger when both the obligatory LRRC8A subunit and the other co-expressed isoform (LRR8C or -E) carried such mutations. Hence, the C-terminal part of the first extracellular loop not only determines VRAC inactivation but might also participate in forming its outer pore. Inactivation of VRACs may involve a closure of the extracellular mouth of the permeation pathway.Volume-regulated anion channels (VRACs) 4 are an important part of the molecular machinery involved in cellular volume homeostasis. They open upon cell swelling and allow for the extrusion of intracellular chloride and osmolytes during regulatory volume decrease (1-3). The current ascribed to VRACs, termed volume-activated anion current (I Cl,vol ), is found in all vertebrate cell types (4). Besides their well described role in cell volume regulation, VRACs have been implicated in many physiological processes, such as cell migration and proliferation, apoptosis, cell cycle maintenance, and release of extracellular signaling molecules (1, 4 -6). Further evidence linked VRACs to several pathologies, such as cancer and ischemic stroke (5, 7). However, the lack of knowledge of the underlying protein(s) forming these channels has greatly hampered further exploration and verification of their suggested physiological and pathophysiological roles. Only recently, LRRC8 (leucine-rich repeat-containing 8) family proteins, which display four putative transmembrane domains and a large C terminus with up to 17 leucine-rich repeats, have been identified as essential components of VRACs (8, 9), and LRRC8 heteromers very likely form the channel pore (6, 8, 10). The physiological importance of VRACs is underscored by the drastically increased lethality of constitutive Lrrc8a KO mice that display multiple tissue abnormalities (11). Mo...

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

“…13,14,20,30 LRRC8 family consists of 5 members denoted as A, B, C, D and E. LRRC8E was found to be one of the essential VSOR components necessary for its rapid inactivation at high depolarizing positive potentials in human colonic tumor HCT116 cells. 13,31 In our experiments, coexpression of LRRC8A with LRRC8E or overexpression of LRRC8E alone did not affect significantly the amplitude of VSOR currents in KCP-4 cells (Fig. 4D).…”

Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels

“…Together with the presence of five different LRRC8 genes, which are often coexpressed in the same cell, a hexameric assembly suggests that there could be a large number of differently composed VRAC channels that may have different properties. Indeed, the LRRC8 subunit composition determines the voltage-dependent inactivation of VRAC currents at non-physiological, inside-positive potentials (Ullrich et al, 2016;Voss et al, 2014), and their single-channel amplitudes (Syeda et al, 2016). Furthermore, the LRRC8D subunit is crucial for the VRAC-mediated cellular uptake of blasticidin S (Lee et al, 2014) and of the anti-cancer drugs cisplatin and carboplatin (Planells-Cases et al, 2015).…”

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

“…However, standard systems like HEK or CHO cells endogenously express VRAC, requiring LRRC8 gene-knockout cell lines for mechanistic investigations 6,15 We have recently found that Xenopus oocytes represent a convenient expression system for LRRC8 proteins. 16 Un-injected oocytes, or oocytes injected with single LRRC8 subunits had no detectable VRAC currents, whereas co-injection of LRRC8A and 8C, 8D or 8E (but not 8B) RNA yielded typical VRAC currents that slowly activated in hypotonic conditions.…”