Structure-function relationships of the LRRC8 subunits and subdomains of the volume-regulated anion channel (VRAC)

Figures

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  Figure 1. Overview of regulatory volume decrease (RVD). The flowchart above outlines the general mechanism of RVD and is illustrated by the diagram below it. This process includes multiple cellular players including P2Y2 receptors (P2Y2R), Ca2+-sensing receptors (CaSR), plasma membrane Ca2+ ATPase (PMCA), cation channels, K+ channels, VRAC, phospholipase C (PLC), inositol triphosphate (IP3), and cyclic adenosine monophosphate (cAMP), the end result of which is an efflux of K+ and Cl− to facilitate the efflux of water and subsequent cell shrinking.

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  Figure 2. Cartoon representation of LRRC8A subunit structure and cross section of VRAC. (A) Each LRRC8A subunit is made up of an intracellular N-terminal coil (NTC, residues 1–22), four transmembrane domains (TMD; residues 23–48, 120–144, 259–286, and 315–345) connected by two extracellular loops (EL, residues 49–119 and 287–314) and two intracellular loops (IL, residues 145–258 and 346–412), followed by 15–17 cytoplasmic leucine rich repeats (residues 413–806; Kasuya et al., 2018). (B) Each VRAC is composed of six LRRC8 subunits. This diagram portrays a cross section of an LRRC8A homomer that arranges to produce a hexameric channel with an extracellular domain (ECD), transmembrane domain (TMD). Intracellular linker domain (ILD), and an extensive leucine rich repeat domain (LRRD).

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  Figure 3. Cartoon representations of LRRC8A homohexameric channels and their symmetries. (A–C) Schematic representations of homohexameric channel symmetries. (A) C2 symmetry wherein three subunits take on a specific steric conformation (orange), and the other three subunits take on another conformation (blue). This is also referred to as a “dimer of trimers.” (B) C3 symmetry wherein two subunits take on the first conformation (orange), two subunits take on a second conformation (blue), and the remaining two subunits take on a third conformation (purple). This is also referred to as a “trimer of dimers.” (C) C6 symmetry, wherein each subunit takes on the same conformation. (D–F) Cryo-EM structures of LRRC8A homomeric channels. (D) Human homohexameric LRRC8A channel (RCSB 5ZSU) at 4.25 Å. C3 symmetry (Kasuya et al., 2018). (E) Human homohexameric LRRC8A channel (RCSB 6DJB) at 4.4 Å. C6 symmetry at the ECD, TMD, and ICD; C3 symmetry at the LRRD (Kefauver et al., 2018). (F) Mouse analog of homohexameric LRRC8A channel (RCSB 6G9O) at 4.25 Å. C6 symmetry at the ECD, TMD, and ICD; C3 symmetry at the LRRD (Deneka et al., 2018).

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  Figure 4. Overview of critical pore-defining amino acid residues of the LRRC8A and LRRC8D subunits as determined by cryo-EM and functional analysis experiments. Critical residues of singular LRRC8A (A) and LRRC8D (B) subunits. Visualization of the constrictions of the pores of LRRC8A homomers (C) and LRRC8D homomers (D). ECD, extracellular domain; TMD, transmembrane domain; ILD, intracellular linker domain; IL, intracellular loop; LRR, leucine rich repeat; LRRD, leucine rich repeat domain.