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. 2025 Feb 20;603(15):4201–4211. doi: 10.1113/JP286189

Recent advances in structural characterization of volume‐regulated anion channels (VRACs)

Erkan Karakas 1,2,✉, Kevin Strange 3, Jerod S Denton 3,4,✉
PMCID: PMC12333906  PMID: 39977537

Abstract

Volume‐regulated anion channels (VRACs) encoded by the LRRC8 gene family play essential roles in diverse and fundamentally important physiological processes in vertebrate cells. The recent determination of high‐resolution cryo‐electron microscopy (cryo‐EM) structures of homomeric and heteromeric LRRC8 channel complexes has created unprecedented opportunities for understanding the molecular basis of VRAC structure, function and pharmacology. Native LRRC8 channels are obligatory heteromers composed of at least one LRRC8A subunit together with one of the other paralogues (LRRC8B‐E) with an unknown stoichiometry. This heteromeric nature of endogenously expressed VRACs and the difficulties associated with controlling the composition and stoichiometry of heterologously expressed LRRC8 channels present considerable experimental challenges. The development of LRRC8 chimeras, which exhibit normal functional and regulatory properties and that can be expressed as homomeric channels, circumvents many of these challenges. In this review, we discuss the recent advances in the structural characterization of LRRC8 channels, with a primary focus on the cryo‐EM structures of one such chimera, created by swapping 25 residues from LRRC8A subunits to LRRC8C subunits and termed as 8C‐8A(IL125).

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Keywords: chimera, cryo‐EM, large‐pore channels, LRRC8, structure, volume‐regulated anion channels, VRAC

Abstract figure legend Native LRRC8 channels are obligatory heteromers comprising LRRC8A and at least one other paralogue (LRRC8B‐E) with an unknown stoichiometry. LRRC8A and LRRC8D homomers form non‐functional hexameric channels or hexameric channels with abnormal functional properties. LRRC8C homomers form non‐functional heptameric channels. Structural analysis of hexameric LRRC8A and LRRC8C heteromeric channels reveal the presence of two distinct arrangements of subunits (shown with full lines), while there are many more potential plausible arrangements of subunits (shown with dashed lines, not all possibilities are shown). LRRC8 chimeras exhibit normal functional and regulatory properties and offer many advantages to studying LRRC8 channels as they yield homomeric channels with defined subunit assembly. The structure of one such chimera, LRRC8C‐LRRC8A(IL125), revealed heptameric arrangements.

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Introduction

Volume‐regulated anion channels (VRACs) are important components of cellular osmoregulation and enable cells to adjust their volume in response to osmotic stress (Jentsch, 2016; Strange et al., 2019). VRACs facilitate the efflux of chloride and organic solutes and thereby mediate cell volume regulation. The identification of leucine‐rich repeat‐containing protein 8A (LRRC8A, also known as SWELL1) as an essential subunit of VRACs represented a transformational milestone for the field and set the stage for a deeper exploration of their diverse roles in cell signalling and physiology (Qiu et al., 2014; Voss et al., 2014). Phylogenetic analysis suggested that LRRC8 channels have high homology to pannexin channels and evolved through a fusion of the genes encoding pannexin and a leucine‐rich repeat‐containing protein (Abascal & Zardoya, 2012).

LRRC8A is an obligatory VRAC subunit and requires the assembly with at least one other paralogue (LRRC8B–E) to reconstitute volume‐regulated channel activity (Qiu et al., 2014; Voss et al., 2014). Subunit heteromultimerization confers distinct functional properties to the channel (Jentsch, 2016; Strange et al., 2019) and determines its selectivity for different substrates. For example, VRACs containing LRRC8C or LRRC8E transport cyclic dinucleotides and play a crucial role in antiviral immunity and T cell function (Concepcion et al., 2022; Lahey et al., 2020; Zhou et al., 2020), whereas channels containing LRRC8D subunits are important for the uptake of platinum‐based oncology drugs (Planells‐Cases et al., 2015). VRACs also transport neurotransmitters and cell signalling molecules that are important for neuronal communication (Schober et al., 2017; Yang et al., 2019).

Recent advances in cryo‐electron microscopy (cryo‐EM) have provided groundbreaking insights into the molecular mechanism underlying VRAC structure and regulation. In this focused review, we discuss the challenges posed by channel heteromeric assembly in studying VRAC structure–function relationships and how novel LRRC8 chimeras can help circumvent many of these challenges.

Structural studies of VRAC/LRRC8 channels

Following the identification of the LRRC8 gene family as the channel‐forming component of VRACs, structural studies offered detailed insights into the channels’ molecular architecture. Research initially focused on homomeric channels to avoid the complexities associated with heteromeric channels. Within a year, four groups published high‐resolution structures of human and mouse LRRC8A homomers (Deneka et al., 2018; Kasuya et al., 2018; Kefauver et al., 2018; Kern et al., 2019), with the structure of human LRRC8D homomers reported shortly thereafter (Nakamura et al., 2020). These structures revealed the molecular architecture of individual subunits and the hexameric channel assembly.

Continued cryo‐EM analysis of LRRC8A homomeric channels has provided valuable insights into the distinct structural and pharmacological characteristics of VRACs (Gunasekar et al., 2022). Deneka et al. (2021) subsequently published a series of cryo‐EM structures of LRRC8A in complex with synthetic nanobodies (termed sybodies) that specifically target the LRRC8A subunits and modulate channel activity allosterically. A recent cryo‐EM study reported the structure of LRRC8A homomers, prepared at high ionic strength conditions (with 350 mM NaCl), revealing the previously unresolved N‐terminal residues of LRRC8A and providing valuable insights into the effects of ionic strength on pore structure (Liu et al., 2023). The readers are encouraged to read other excellent reviews discussing in greater detail insights gained from these studies (Kasuya & Nureki, 2022; Sawicka & Dutzler, 2022; Strange et al., 2019; Syrjanen et al., 2021).

Structure of VRAC/LRRC8 subunits

LRRC8 subunits are organized into two distinct regions: the pore‐forming region at the N‐terminal half and the leucine‐rich repeat domain (LRRD) at the C‐terminal half (Fig. 1). As predicted from sequence analysis, the structure of the pore region is highly similar to that of pannexin. It is composed of the extracellular domain (ECD), the transmembrane domain (TMD) and the intracellular domain (ICD), which together form an oligomeric assembly arranged in a ring‐like structure around an axis perpendicular to the membrane plane (Fig. 1). The first 14 N‐terminal residues in most structures are disordered and not resolved. The TMD consists of four transmembrane helices: TM1, which primarily lines the pore; TM3 and TM4, which face the lipid bilayer; and TM2, which interfaces with TM1 and TM4 of adjacent subunits. The first extracellular loop (EL1) between TM1 and TM2 features a β‐strand (EL1‐β1) and an α‐helix (EL1‐H1) with a largely disordered intervening region. The second extracellular loop (EL2) between TM3 and TM4 contains two β‐strands (EL2‐β1 and EL2‐β2) linked by a short, well‐defined loop, forming a β‐sheet with EL1‐β1. Two disulphide bonds stabilize the β‐sheet and α‐helix in both LRRC8A and LRRC8D, with a third disulphide bond within the region connecting EL1‐β1 and EL1‐H1 exclusive to LRRC8A structures. The cysteine residues forming the disulphide bonds within the β‐sheet and α‐helix are conserved across all five subtypes, but the cysteines forming the third disulphide bond are only present in LRRC8A and LRRC8B. The ICD comprises two loops: IL1, which connects TM2 to TM3 and includes three α‐helices (IL1‐H1, IL1‐H2 and IL1‐H3); and IL2, which bridges TM4 to the LRRD and consists of four α‐helices (IL2‐H1, IL2‐H2, IL2‐H3 and IL2‐H4). The LRRDs of the LRRC8 subunits feature a curved solenoid structure, beginning with an N‐terminal α‐helix, continuing with a series of 15–17 leucine‐rich repeats (LRR1‐15) and concluding with a C‐terminal segment composed of three α‐helices.

Figure 1. Structure of the LRRC8A subunit.

Figure 1

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Ribbon representation of the LRRC8A subunit (PDB ID: 7XZH). Rainbow colouring from blue to red indicates the N‐ to C‐terminal positions of the residues. The N‐terminal fragment only observed in this structure is shown transparent.

Structure of homomeric VRAC/LRRC8 channels

Homomeric LRRC8A and LRRC8D channels typically form symmetric oligomers with well‐resolved subunit interfaces (Deneka et al., 2021, 2018; Gunasekar et al., 2022; Kasuya et al., 2018; Kefauver et al., 2018; Kern et al., 2019; Liu et al., 2023; Nakamura et al., 2020). In LRRC8A, a symmetry mismatch is observed, where the pore region has six‐fold or pseudo‐six‐fold symmetry, and the LRRDs are arranged as a trimer of dimers with three‐fold symmetry (Fig. 2A ). The interfaces between dimers within the LRRDs are extensive and consistently preserved across various structures, whereas the trimeric interfaces are smaller and more variable, resulting in structures with varying degrees of heterogeneity with the LRRD arrangement (Deneka et al., 2021, 2018; Gunasekar et al., 2022; Kasuya et al., 2018; Kefauver et al., 2018; Kern et al., 2019; Liu et al., 2023). In contrast, the LRRC8D structure presents a two‐fold symmetric arrangement, distinctly different from that of LRRC8A (Nakamura et al., 2020).

Figure 2. Cryo‐EM structures of VRACs.

Figure 2

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Ribbon representation of the cryo‐EM structure of: A, LRRC8A homomer (PDB ID: 7XZH); B, 8C‐8A(IL125) chimera (PDB ID: 8DXN); C, LRRC8C homomer (PDB ID:8B40); D, LRRC8A:LRRC8C (4:2) heteromeric channels (PDB ID: 8B41); and E, LRRC8A:LRRC8C (5:1) heteromeric channels (PDB ID: 8DS3). The LRRC8A subunits are coloured light pink. The LRRC8C and 8C‐8A(IL125) subunits are coloured cyan. The structures at the bottom panels are viewed from the cytoplasmic side focusing on the ICD and omitting the LRRDs. The unmodelled LRRDs indicate that they were highly heterogeneous resulting in weak cryo‐EM maps.

Function and structure of homomeric VRAC/LRRC8 chimeric channels

While structural insights from homomeric channels have been invaluable for understanding the structural features of VRACs, directly correlating these features with VRAC activity has been challenging. Homomeric channels do not exhibit the physiological properties of native VRACs, which function as obligate heteromers (Yamada et al., 2021). Homomeric LRRC8C and LRRC8D channels are not expressed at the cell surface and therefore have not been functionally characterized (Voss et al., 2014). Homomeric LRRC8A channels are expressed at the cell membrane but are poorly activated only by extreme cell swelling or reductions in intracellular ionic strength (Yamada et al., 2021). Homomeric LRRC8A currents also exhibit grossly abnormal 4‐[(2‐Butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid (DCPIB) pharmacology (Yamada et al., 2021).

Yamada & Strange (2018) discovered that substituting 25 amino acids from the first intracellular loop of LRRC8C with the corresponding sequence from LRRC8A conferred normal functionality to LRRC8C channels, endowing them with physiological and pharmacological properties akin to native heteromeric channels (Fig. 3). This chimera, termed 8C‐8A(IL125), can be expressed as a homomeric channel and, therefore, circumvents many of the experimental challenges associated with heteromeric LRRC8 channels.

Figure 3. Functional characteristics of the 8C‐8A(IL125) chimera.

Figure 3

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A and B, schematic diagrams of the individual subunits (left) and time course of resting, swelling‐activated and shrinkage‐inactivated currents in LRRC8−/− HCT116 cells (right) co‐expressing the LRRC8A and LRRC8C (A) or expressing 8C‐8A(IL125) chimera (B). Figures are reproduced from Takahashi et al. (2023) and Yamada & Strange (2018).

Our structural analysis of the 8C‐8A(IL125) chimera revealed important similarities and differences compared to the homomeric LRRC8A and LRRC8D channels (Takahashi et al., 2023). Whereas homomeric LRRC8A and LRRC8D are hexamers, the 8C‐8A(IL125) chimera forms heptameric assemblies, similar to that of homologous pannexin channels (Fig. 2B ) (Takahashi et al., 2023). Although not forming functional channels when expressed alone, unmodified LRRC8C subunits were also shown to assemble as heptamers (Fig. 2C ) (Rutz et al., 2023). This indicates that LRRC8 subunits can assemble in multiple oligomeric states.

Structure of VRAC/LRRC8 heteromeric channels

More recently, cryo‐EM structures of heteromeric channels composed of LRRC8A and LRRC8C have been determined (Kern et al., 2023; Rutz et al., 2023) and offered clues to how channel stoichiometry may affect channel gating (Fig. 2D, E ).

Working with heteromeric channels presents significant challenges at different stages of the experimental process. The first challenge involves obtaining homogeneous protein samples with a defined stoichiometric assembly. Assuming a hexameric arrangement, there can be 12 distinct assemblies of LRRC8A/C heteromers, in addition to potential homomeric LRRC8A and LRRC8C channels, when these subunits are co‐expressed. The heterogeneity could be even greater if heteromeric channels assemble as heptamers, similar to the 8C‐8A(IL125) chimera and LRRC8C homomers. Perhaps fortuitously, the expression systems used by these groups resulted in the purification of homogeneous protein samples with a predominant stoichiometry. Curiously, the stoichiometry differed between the studies. Rutz et al. (2023) performed large‐scale expression using HEK293 cells transfected with equimolar concentrations of plasmids encoding LRRC8A and LRRC8C, yielding protein samples with 4:2 LRRC8A:LRRC8C ratio. Kern et al. (2023). expressed the protein using Sf9 insect cells and obtained protein samples with predominantly 5:1 LRRC8A:LRRC8C stoichiometry.

Another technical challenge is differentiating the LRRC8A and LRRC8C subunits during particle alignment, as these subunits are likely to appear identical during initial data processing, creating pseudo‐symmetry that prohibits high‐resolution structure determination. To overcome this obstacle, the two studies employed distinct approaches. Rutz et al. (2023) mixed the protein samples with sybodies (synthetic nanobodies) specific to LRRC8A subunit for cryo‐EM experiments. The additional density resulting from sybodies attached to LRRC8A acted as fiducial markers, aiding in the alignment of particles during data processing and enabling unambiguous identification of the LRRC8A and LRRC8C subunits in the structure. Kern et al. (2023) took a different approach, genetically incorporating the BRIL domain (b562RIL; an engineered variant of cytochrome b562a) and replacing a disordered loop in the first extracellular loop of LRRC8A. The size of the BRIL domain was further increased by adding anti‐BRIL antibody fragments (Fabs) and anti‐Fab nanobodies.

Oligomeric assembly and pore structure

The stoichiometric arrangement of VRACs has been a subject of intensive investigation, with earlier biochemical studies suggesting a hexameric composition as a minimum (Gaitán‐Peñas et al., 2016; Syeda et al., 2016). Homomeric LRRC8A and LRRC8D channels are assembled as hexamers (Fig. 3A ) (Deneka et al., 2021, 2018; Gunasekar et al., 2022; Kasuya et al., 2018; Kefauver et al., 2018; Kern et al., 2019; Liu et al., 2023; Nakamura et al., 2020). Recent structural studies of heteromeric LRRC8A–LRRC8C channels also revealed hexameric assemblies, albeit with varying stoichiometries, including 4:2 and 5:1 ratios of LRRC8A to LRRC8C (Fig. 2D, E ) (Kern et al., 2023; Rutz et al., 2023). Rutz et al. (2023) utilized quantitative mass spectroscopy to analyse heteromeric channels purified from wild‐type and LRRC8B,D,E knockout HEK293 cells, reporting LRRC8A to LRRC8C ratios of 1.8:1 and 2.9:1, respectively. They also noted a 2:1 ratio when LRRC8A and LRRC8C subunits were overexpressed at equimolar levels, with a slight deviation in favour of LRRC8C when its DNA was used at threefold higher levels (Rutz et al., 2023). Using fluorescent photobleaching experiments, Kern et al. (2023) observed more variable stoichiometry and proposed that channels with multiple stoichiometries can form depending on the relative expression levels of the subunits.

Adding to the complexity, structures of the homomeric 8C‐8A(IL125) chimera and LRRC8C channels have been identified as heptamers (Fig. 2B, C ) (Rutz et al., 2023; Takahashi et al., 2023). This suggests that the subunit identity and ratio may influence the oligomeric state, with an increased presence of LRRC8C subunits favouring a heptameric arrangement. Modulating subunit oligomeric state could function as a cellular regulatory mechanism to adjust pore size for accommodating permeants of varying dimensions, as heptameric channels will inherently possess larger pores than their hexameric counterparts (Fig. 4). For example, the 8C‐8A(IL125) chimera demonstrates enhanced permeability to larger anions such as glutathione and lactobionate when compared to homomeric LRRC8A channels, consistent with a larger pore diameter (Takahashi et al., 2023).

Figure 4. Comparison of the accessible pore sizes.

Figure 4

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The solvent‐accessible surface representation of the constriction site at the ECD as viewed from cytoplasm for the structures: A, LRRC8A homomer (PDB ID: 5ZSU); B, LRRC8A homomer (PDB ID: 7XZH); C, LRRC8A:LRRC8C (5:1) heteromeric channels (PDB ID: 8DS3); D, LRRC8A:LRRC8C (4:2) heteromeric channels (PDB ID: 8B41); and E, 8C‐8A(IL125) chimera (PDB ID: 8DXN). The LRRC8A subunits are coloured light pink. LRRC8C and 8C‐8A(IL125) subunits are coloured cyan. Pore size is substantially larger in the heptameric 8C‐8A(IL125) chimera.

The narrowest point within the VRAC pore is situated at the ECD. Homomeric LRRC8A structures feature arginine 103 (R103) side chains at this position that converge towards the centre and form a positively charged barrier along the permeation pathway. The pore sizes appear variable due to the diverse rotamers that arginine residues adopt in different structures (Fig. 4A, B ). In heteromeric LRRC8A–LRRC8C structures, this arginine is replaced by leucine residues (L105) from LRRC8C subunits, leading to a slightly different pore size and a modified electrostatic surface charge (Fig. 4C, D ). The equivalent positions are occupied by arginine in LRRC8B, phenylalanine in LRRC8D and leucine in LRRC8E, suggesting that pore size and electrostatic properties vary with subunit composition and stoichiometry, influencing permeant selectivity.

Despite these variations in amino acid residues at the constriction site, the observed structural conformations across different subunits suggest that the pore size is barely large enough to pass small anions such as chloride, irrespective of subunit composition. To accommodate larger anions at this constriction site, more substantial conformational changes would be required to sufficiently expand the pore, particularly in hexameric channels. Alternatively, it is possible and even likely that at least some native VRACs comprise heptameric or higher‐order oligomeric structures. Estimates from electrophysiological studies of native VRAC pore radius range from 6 to 7 Å (Droogmans et al., 1999; Ternovsky et al., 2004), which aligns well with a pore radius of 4.7 Å observed for heptameric 8C‐8A(IL125) channels but is in striking contrast to a pore radius of 2.0–3.5 Å for homohexameric LRRC8A and LRRC8D and heterohexameric LRRC8A/LRRC8C channels (Deneka et al., 2018; Droogmans et al., 1999; Gunasekar et al., 2022; Kasuya et al., 2018; Kefauver et al., 2018; Kern et al., 2023, 2019; Liu et al., 2023; Nakamura et al., 2020; Rutz et al., 2023; Takahashi et al., 2023; Ternovsky et al., 2004). Hypothetical models of LRRC8A and LRRC8D channels containing seven subunits have pore radii of 4.7 and 7.1 Å, respectively, indicating that oligomeric state has a direct impact on pore size and probably on channel transport properties (Takahashi et al., 2023). A critical outstanding question is whether native VRACs exist as hexamers or assemble into high‐order structures.

Structural heterogeneity of LRRC8 subunits

We observed a greater degree of conformational heterogeneity in the 8C‐8A(IL125) chimera structures compared to LRRC8A and LRRC8D (Takahashi et al., 2023). Through 3D classification, five distinct conformations were identified, with poorly resolved LRRD densities indicating the presence of additional, unresolved conformations (Fig. 5). Unlike the symmetric structures of LRRC8A and LRRC8D, the 8C‐8A(IL125) chimera displays asymmetry, except for the ECD, which maintains a sevenfold symmetry. The LRRDs exhibit the greatest variability in subunit arrangement within these conformations, and considerable flexibility is also observed in the ICD and TMD (Takahashi et al., 2023). We suspect that increased flexibility plays an important role in channel regulation.

Figure 5. Structural heterogeneity of LRRDs.

Figure 5

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A and B, Unsharpened cryo‐EM maps of the five classes obtained from a single dataset of the 8C‐8A(IL125) chimera. Each subunit is coloured differently. The LRRDs exhibit distinct arrangements across the classes. Figures are reproduced from Takahashi et al., 2023

In contrast to the 8C‐8A(IL125) chimera, homomeric LRRC8C channels exhibit less heterogeneity and predominantly form symmetric heptameric structures (Rutz et al., 2023). The intracellular loop IL125 from LRRC8A, which imparts functionality to LRRC8C in the chimera, may be responsible for the observed increase in flexibility or trafficking of the channels to the plasma membrane (Takahashi et al., 2023; Yamada & Strange, 2018). However, the IL125 loop itself is not resolved in the cryo‐EM maps, leaving its precise role in channel regulation an open question for future research (Takahashi et al., 2023). The loop's function may be limited to facilitating channel trafficking to the plasma membrane, or it may play a more dynamic role in modulating channel activity, potentially through interactions with the LRRDs.

Lipid block and gating

A central question in the study of ‘large‐pore’ channels such as VRAC is the location and mechanism of pore gating. A provocative hypothesis is that the pore is occluded by lipids in the closed state, and pore opening involves a rearrangement of these lipids during gating to create a hydrophilic conduction pathway that can be permeated by chloride. Supporting this hypothesis, cryo‐EM maps of other large‐pore channels including pannexins, innexins and calcium homeostasis modulator (CALHM) have revealed pronounced densities within the pores, resembling lipid bilayers that could block permeation (Burendei et al., 2020; Drożdżyk et al., 2020; Kuzuya et al., 2022; Syrjanen et al., 2021, 2020).

Cryo‐EM maps of the 8C‐8A(IL125) chimera also display two layers of strong density within the pore (Fig. 6A ) (Takahashi et al., 2023). These align well with the boundaries of the phosphate moieties of the phospholipid bilayer surrounding the TMD and are likely to arise from the phospholipids filling the pore. Furthermore, both hydrophobicity and surface charge profiles within the pore appear favourable to accommodate a lipid bilayer, where the polar head groups would interact with the charged residues on cytoplasmic and extracellular sides of the TMD and the acyl chains would interact with the mostly hydrophobic surface of the space in between in the TMD. In this model, lipids fill the pore entirely, and their reorganization is necessary to enable permeant passage.

Figure 6. Lipid interaction of the pore region.

Figure 6

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A, a sliced view of the unsharpened cryo‐EM map of the 8C‐8A(IL125) chimera focusing on the lipid‐like density blocking the permeation pathway. Figure is reproduced from Takahashi et al., 2023 B, ribbon representation of the 8C‐8A(IL125) structure (PDB ID: 8DXN) along with the lipid‐like cryo‐EM maps (red) penetrating through the subunit interfaces. C, surface representation of the LRRC8A:LRRC8C (5:1) heteromeric channels (PDB ID: 8DS3) along with modelled lipid molecules both penetrating through the subunit interfaces and blocking the permeation pathway.

All available structures indicate large gaps between the subunits within the TMD, and these spaces are filled with cryo‐EM densities that correspond to the size and shape of phospholipids (Fig. 6B, C ). Some of these densities are well resolved, showing polar head groups of the lipids interacting with basic residues on the outer surface of the TMD and the acyl chains protruding toward the inner surface of the pore. The dynamic spacings between subunits, especially noted in 8C‐8A(IL125), imply that lipid rearrangements may accompany conformational changes in the channel. Indeed, the clarity of the lipid densities diminishes as the separation between the subunits increases (Takahashi et al., 2023).

Further support for this lipid‐mediated gating model comes from heteromeric LRRC8A‐LRRC8C structure reported by Kern et al. (2023), where lipid‐like densities block the pore (Fig. 6C ). These densities are more distinct in the smaller hexameric channel, showing three phospholipids at the extracellular TMD side, reducing the solvent‐accessible path and rendering the channel impermeable. Mutational analysis targeting residues interacting with lipid acyl chains and glycerol backbones has shed light on gating mechanics. The T48D mutation in LRRC8A, which introduces negative charges, results in an increase in the basal activity of LRRC8A‐T48D/LRRC8C channels and disrupts the density of pore‐blocking lipids in LRRC8A‐T48D/LRRC8C cryo‐EM maps. This suggests that the mutation impairs lipid binding, maintaining an open pore (Kern et al., 2023). Similar but considerably weaker densities were also observed in LRRC8A–LRRC8C channels reported by Rutz et al. (2023), although the authors were cautious to call them lipids. However, considering that LRRC8 channels evolved from pannexins (Abascal & Zardoya, 2012), which appear to be gated by pore‐resident lipids (Kuzuya et al., 2022), we hypothesize that it is likely that VRACs are also gated by lipids.

A critical aspect to consider in the structural analysis of VRACs is that the N‐termini of the subunits remain unresolved in structures exhibiting lipid‐like densities within the pore (Rutz et al., 2023; Takahashi et al., 2023). Conversely, in structures where the N‐terminal residues are resolved, they occupy the positions hypothesized for lipid molecules (Liu et al., 2023; Nakamura et al., 2020). This suggests a possible interplay between the lipids and the N‐terminal residues, which could significantly influence the gating properties of the channels. In support of this notion, Liu et al. (2023) recently demonstrated that the structure of the N‐terminus is modulated by ionic strength, suggesting that it may modulate cell swelling‐induced activation and cell shrinkage‐induced inactivation of LRRC8 channels. Further investigations are necessary to elucidate the precise nature of the N‐terminus in channel gating.

Mechanism of DCPIB block

Emerging data from gene knockout studies have uncovered new and potentially druggable functional roles of VRAC in numerous human pathologies, from type 2 diabetes and hypertension to ischaemic stroke injury and pain. The molecular pharmacology of VRAC/LRRC8 channels is poorly developed and consists mainly of low‐potency, non‐specific inhibitors. The best‐in‐class inhibitor is DCPIB, which inhibits native VRAC at single‐micromolar concentrations, but has multiple off‐target effects on ion channels, transporters and mitochondrial respiration (reviewed in Figueroa & Denton, 2022). Structural insights into VRAC pharmacology were gained through the structures of LRRC8A, first in complex with DCPIB and then with SN‐407, a DCPIB analogue (Gunasekar et al., 2022; Kern et al., 2019). DCPIB and SN‐407 are moderately potent inhibitors of VRACs. The structures revealed that the inhibitors act as a plug in the extracellular entrance of the pore and the carboxylic acid moiety of the inhibitor is coordinated by R103 at the constriction site. However, the LRRC8A homomeric channels exhibit grossly abnormal DCPIB pharmacology (Yamada et al., 2021), including weak and voltage‐dependent channel inhibition, which is not observed in native VRAC. Furthermore, mutation of LRRC8A‐R103 to phenylalanine (R103F) abrogates the block of homomeric LRRC8A channels by DCPIB but has no effect on heteromeric LRRC8A‐R103F/LRRC8C channels (Yamada et al., 2021). This indicates that the mechanism of DCPIB inhibition of homohexameric LRRC8A is fundamentally different from that of native VRAC. Detailed analysis of the DCPIB mechanism of action awaits further study. We propose that LRRC8 chimeras, such as the 8C‐8A(IL125), could potentially become valuable tools for elucidating the mechanism of action of DCPIB and aiding in the development of new drugs targeting VRAC, given their ability to simplify the study of channel structure, function and regulation while maintaining native‐like physiological and pharmacological properties (Yamada & Strange, 2018; Yamada et al., 2021).

Future perspectives

The decade since the identification of the LRRC8 gene family as the component of VRACs has seen remarkable progress in understanding the molecular structure, regulation and pharmacology of LRRC8 channels. Several pressing questions remain to be addressed: First, how many subunits do native VRACs contain, and what is the stoichiometry and subunit arrangement of native channels? The current best available evidence suggests that they are heteromers containing six, seven or possibly even more subunits (Gaitán‐Peñas et al., 2016; Kern et al., 2023; Rutz et al., 2023; Syeda et al., 2016; Takahashi et al., 2023). Second, how do LRRC8 channels transduce changes in cell volume or membrane tension into the gating of the channel pore? Emerging evidence indicates that conformational changes involving cytoplasmic LRRDs and pore lipids are involved in some as‐yet‐to‐be determined mechanisms (Deneka et al., 2021). The variable, indeterminate and experimentally uncontrollable subunit composition and stoichiometry of LRRC8 channels complicate the molecular analysis of VRAC function and gating.

Use of the 8C‐8A(IL125) chimera exemplifies the significant experimental advantages of using chimeric constructs with native‐like functional properties to circumvent many of these complications by enabling the structure–function analysis of VRACs with defined composition and normal physiological properties. Yamada and Strange (2018) reported a number of other chimeric constructs that also exhibit normal functional and regulatory properties, further expanding the toolkit available for VRAC research. These chimeras provide a unique opportunity to dissect the specific contributions of different LRRC8 subunits and regions to channel function. Similar to LRRC8C subunits, incorporation of the LRRC8A IL1 confers functionality to LRRC8D and LRRC8E subunits, facilitating their structural and functional characterizations.

Finally, do LRRC8/VRAC channels represent druggable targets for diseases, and can potent and selective inhibitors and activators be developed? Ongoing high‐throughput screening campaigns hold tremendous promise for rapidly advancing the molecular pharmacology of the LRRC8/VRAC channel family (Chu et al., 2023; Figueroa et al., 2019; Figueroa & Denton, 2021, 2022). Chimeric channels such as the 8C‐8A(IL125), which can be expressed as homomers, simplify the investigation of VRAC pharmacology. The ability to produce homomeric channels with defined subunit compositions eliminates the variability associated with heteromeric assemblies, allowing for more precise drug screening and characterization of channel inhibitors and activators.

Additional information

Competing interests

Kevin Strange is founder and CEO of Revidia Therapeutics, Inc.

Author contributions

All authors contributed to the manuscript preparation and approved the final version of the manuscript.

Funding

Dr Karakas’ lab is supported by grants from the National Institute of Health (R01GM141251). Dr Denton's lab is supported by a grant from the National Institution of health (R01DK051610).

Supporting information

Peer Review History

TJP-603-4201-s001.pdf (240.3KB, pdf)

Biographies

Erkan Karakas is an Associate Professor at Vanderbilt University. He earned his BSc from Middle East Technical University in Turkey, his PhD from Stony Brook University in New York and completed postdoctoral research at Cold Spring Harbor Laboratory, New York. His lab focuses on the structural biology of ion channels and their regulatory mechanisms.

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Kevin Strange is Founder, CEO and President of Revidia Therapeutics, Inc., a regenerative medicine drug development company, and Professor of Anesthesiology at Vanderbilt University Medical Center. His laboratory defined many of the basic functional properties of native VRACs and developed and characterized multiple LRRC8 chimeras.

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Jerod Denton is a Professor of Anesthesiology and Pharmacology at Vanderbilt University Medical Center and Director of Ion Channel Pharmacology at the Warren Center for Neuroscience Drug Discovery. The major focus of his lab is on the molecular pharmacology of ion channels.

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Handling Editors: Laura Bennet & Jorge Contreras

The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP286189#support‐information‐section).

Contributor Information

Erkan Karakas, Email: erkan.karakas@vanderbilt.edu.

Jerod S. Denton, Email: jerod.s.denton@vumc.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Peer Review History

TJP-603-4201-s001.pdf (240.3KB, pdf)

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