Structure of a LRRC8 chimera with physiologically relevant properties reveals heptameric assembly and pore-blocking lipids

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Volume-regulated anion channels (VRACs) mediate Cl - and organic solute efflux from vertebrate cells and are essential for cell volume homeostasis. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a hexameric structure. However, these channels are either non-functional or exhibit abnormal functional properties limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with physiologically relevant functional properties. We demonstrate here that the LRRC8C-LRRC8A(IL1 25 ) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Membrane lipids are a key structural element of the channel and are located between subunits and occluding the channel pore. Our results suggest that native VRAC/LRRC8 channels are heptamers and that associated lipids are likely essential for normal channel gating and regulation.

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  1. eLife assessment

    This paper, which provides useful information on the assembly of volume-regulated anions channels formed by LRRC8 proteins, will be of interest scientists in the field of ion channels. The authors report the structure of a LRRC8C-LRRC8A chimera with native functional properties as a heptameric complex with a lipid-filled pore. This is very interesting and well-presented work, but the evidence supporting the physiological relevance of the heptameric assembly and the hypothesized role of lipids is still incomplete.

  2. Reviewer #1 (Public Review):

    This manuscript by the Karakas lab reports on new structures of the volume regulated LRRC8 anion channels. These ubiquitously expressed channels play key roles in cell volume regulation and in allowing efflux of organic osmolytes, neurotransmitters, and drugs. In addition to regulating cell volume LRRC8 channels might play roles in signal transduction, cell migration, apoptosis, tumor drug resistance, and stroke. Thus, elucidating their architecture and structure is of critical importance. LRRC8 channels are obligate multimers of variable stoichiometry, with the LRRC8A subunit being absolutely required for assembly of functional channels. Structures of homomeric LRRC8A and LRRC8D channels revealed a hexameric assembly with closed pores. However, the functional properties of these homomeric channels differ from those of recorded in cells, raising questions on the physiological relevance of these conformations. The authors here determine the structure of a LRRC8C-LRRC8A chimera (termed 8C-8A(IL125)) with functional properties that closely resemble those of native channels. Remarkably, the 8C-8A(IL125) chimera assembles as a heptamer with a large pore. Unexpectedly, in the structures the channel's pore is occupied by density that could correspond to lipids.

  3. Reviewer #2 (Public Review):

    Volume-regulated anion channels (VRACs), comprised of the LRRC8 family of proteins, play important roles in cell volume regulation. Physiological LRRC8 channels are heteromeric assemblies of LRRC8A and LRRC8B-LRRC8E subunits. Previous structural studies have focused on homomeric channels, which do not recapitulate functional properties of native heteromeric channels. Thus, the molecular basis of physiological VRAC assembly and function remains unknown. In this study, Takahashi and colleagues present the single-particle cryo-electron microscopy structure of a functional LRRC8 chimera, which is composed of LRRC8C and a swapped intracellular loop from LRRC8A. Surprisingly, the chimeric channel forms a heptamer, in sharp contrast to the previously reported hexamers of homomeric and heteromeric LRRC8 channels. The findings of the chimeric channel are interesting. However, the physiological implication of this chimera is unclear, and the proposal that native LRRC8 channels are heptamers is not well supported.

  4. Reviewer #3 (Public Review):

    This manuscript by Takahashi et al., reveals the structure of a chimeric VRAC channel composed by the LRRC8C and a short domain corresponding to the intracellular loop of LRRC8A. Homomeric LRRC8C channels are not functional but this chimera has been shown to "rescue" the functional and pharmacological properties of heteromeric VRAC channels. The authors obtained the Cryo-EM structure for this chimera, which provide some interesting insights about these channels. The major finding of this work is that the channel is asymmetrically formed by 7 protomers, with associated lipid-like densities that are proposed to play a role in gating. Unfortunately, critical domains of this structure could not be solved, which limit the interpretations of this new work. These missing domains include the entire LRRD, the N-terminus and the first intracellular loop containing the 25 amino acids incorporated from the LRRC8A. While this work is very interesting, the data presented are not enough to support the author claims. Particularly, the idea that the 'lipid blocked pore' is associated with gating.