Abstract
Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.
Highlights
Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC)
When expressed in Sf9 insect cells from Spodoptera frugiperda, which lacks endogenous LRRC8 genes[13], fluorescence-detection sizeexclusion chromatography[24] of the C-terminally EGFP-tagged human LRRC8D (HsLRRC8D; the “Hs” refers to Homo sapiens) protein showed a sharp and monodisperse peak with a similar elution volume to that of the human LRRC8A (HsLRRC8A) protein, with structures determined by cryo-electron microscopy (cryo-EM) single-particle analysis[15,16], indicating that the HsLRRC8D protein is a promising candidate for structural analysis (Supplementary Fig. 1a)
A mass spectrometry analysis of the purified HsLRRC8D protein sample from Sf9 insect cells confirmed that the sample contains no LRRC8 isoforms other than HsLRRC8D and that the HsLRRC8D protein exists as a homomer (Supplementary Fig. 1b, c; Supplementary Data 1)
Summary
Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). The isoform combinations forming LRRC8 hetero-hexamers underlie the functional diversity of the channel properties, including open probability[7], gating kinetics[4,9], and substrate specificity[8,10]. Recent electrophysiological analyses demonstrated that the first several N-terminal residues preceding the TM1 helix[16,23] and the first extracellular loop (EL1) between the TM1 and TM2 helices[9,12] are involved in the diversity of channel properties, including ion permeability and selectivity as well as the kinetics of inactivation. We determined the structure of the human LRRC8D homo-hexamer, including the N-terminal region, by cryo-EM single-particle analysis, and conducted a structure-based electrophysiological analysis to investigate the mechanism underlying the contribution of the LRRC8D isoform to the LRRC8 protein function
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