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Structural and functional characterization of the bestrophin-2 anion channel

Nature Structural & Molecular Biology volume 27, pages 382–391 (2020)Cite this article

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Abstract

The bestrophin family of calcium (Ca2+)-activated chloride (Cl−) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca2+, comprises four members in mammals (bestrophin 1–4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca2+ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca2+, it has substantial Ca2+-independent activity for Cl−, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca2+ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.

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Fig. 1: Cryo-EM structure of bBest2 and comparison to cBest1 and KpBest.
Fig. 2: Ca2+-dependent Cl− current mediated by Best1 and Best2.
Fig. 3: Ca2+-dependence and ion selectivity of bBest2.
Fig. 4: The role of the neck and aperture in Ca2+-dependent gating of Cl−.
Fig. 5: The role of the neck and aperture in Ca2+-dependent gating of CH3SO3−.
Fig. 6: The role of the neck and aperture in Ca2+-dependent gating of I−.
Fig. 7: Critical residues for the ion selectivity of bBest2.

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Data availability

Atomic models are available through the Protein Data Bank with accessions codes 6VX5, 6VX6, 6VX7, 6VX8 and 6VX9; cryo-EM reconstructions are available through the Electron Microscopy Data Bank with accession codes EMD-21430, EMD-21431, EMD-21432, EMD-21433 and EMD-21434.

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Acknowledgements

We thank the Center on Membrane Protein Production and Analysis (New York Structural Biology Center, supported by the National Institutes of Health (NIH) grant no. GM116799) and A. Sobolevsky for help on mammalian bestrophin screening; E. Eng for help on collecting cryo-EM data and R. Grassucci for assistance with microscope operation for initial screening experiments. Cryo-EM data were collected at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy (New York Structural Biology Center), supported by grants from the Simons Foundation (no. 349247), NYSTAR and NIH (no. GM103310). A.P.O. was supported by NIH grant no. (F31) EY030763, Y.S. was supported by the National Key R&D Program of China (2017YFE0103400), W.A.H. was supported by NIH grant no. GM107462 and T.Y. was supported by NIH grant nos. EY025290 and GM127652 and Columbia University start-up funding.

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  1. These authors contributed equally: Aaron P. Owji, Qingqing Zhao.

Authors and Affiliations

  1. Department of Pharmacology, Columbia University, New York, NY, USA

    Aaron P. Owji

  2. Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY, USA

    Qingqing Zhao, Changyi Ji, Alec Kittredge, Austin Hopiavuori, Nancy Ward & Tingting Yang

  3. Eye Center, Renmin Hospital of Wuhan University, Wuhan, China

    Qingqing Zhao & Yin Shen

  4. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Ziao Fu & Wayne A. Hendrickson

  5. Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA

    Oliver B. Clarke & Wayne A. Hendrickson

  6. Department of Ophthalmology, Columbia University, New York, NY, USA

    Yu Zhang & Tingting Yang

  7. New York Structural Biology Center, New York, NY, USA

    Wayne A. Hendrickson

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Contributions

A.P.O. designed research, performed protein purification and cryo-EM experiments, analyzed data, made figures and helped with writing the paper. Q.Z. and C.J. performed and analyzed patch clamp recordings, A.K. made constructs and purified proteins, A.H. maintained cells, Z.F. helped with collecting and analyzing cryo-EM data and N.W. made viruses. O.B.C. helped with cryo-EM data processing. Y.S. supervised Q.Z. and edited the manuscript. Y.Z. screened and purified proteins and wrote the paper. W.A.H. designed research and analyzed data. T.Y. designed research, analyzed data, made figures and wrote the paper.

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Correspondence to Yin Shen, Yu Zhang, Wayne A. Hendrickson or Tingting Yang.

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Peer review information Katarzyna Marcinkiewicz and Ines Chen were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Structure determination of bBest2 with 250 nM Ca2+.

a, b, FSC curve for masked and unmasked map generated by cryoSPARC v2 non-uniform refinement for Ca2+-bound (a) and Ca2+-unbound (b) states. c, d, Local resolution estimation calculated by blocres as implemented in cryoSPARC v2 for Ca2+-bound (c) and Ca2+-unbound (d) states. e, Viewing angles for particles contributing to final map as implemented by pyem star2bild script for Ca2+-bound state. f, Analysis of sphericity of final maps by 3DFSC server to assess directional anisotropy of specimens with slightly preferred orientation for Ca2+-bound state. g, h, The same format for the Ca2+-unbound state. Blue histogram indicates percentage of per angle FSC. Red solid line indicates directional FSC and green dashed line indicates + /− 1 standard deviation from mean of directional FSC. i, j, Map v. Model FSC curve with and without mask as implemented by Phenix.validation package for Ca2+-bound (i) and Ca2+-unbound (j) states.

Extended Data Fig. 2 Structure determination of bBest2 with EGTA only.

In the same format as Extended Data Fig. 2. Left (a, c, e, f, i), Ca2+-unbound state 1 (N-terminus partially disordered); right (b, d, g, h, j), Ca2+-unbound state 2 (N-terminus completely disordered).

Extended Data Fig. 3 Structure determination of bBest2 with 5 mM Ca2+.

In the same format as Extended Data Figs. 2 and 3. a, FSC curve generated in cryoSPARC. b, Local resolution from blocres. c, Viewing angles for particles contributing to final map. d, Analysis of sphericity. e, Map v. Model FSC curve with and without mask.

Extended Data Fig. 4 Representative cryo-EM density for the two bBest2 cryo-EM structures with 250 nM Ca2+.

a, Architecture of an individual bBest2 protomer (left) and cBest1 protomer (4RDQ, right), both Ca2+-bound and color-coded by segments in accordance with Supplementary Figure 1. b–h, Representative map densities for indicated regions are shown with the corresponding atomic model. The Ca2+-bound state (ordered N-terminus) and Ca2+-unbound state (completely disordered N-terminus) structures are shown in orange (left) and blue (right), respectively. Green dot, a Ca2+ ion. i, j, Stereo images of bBest2, presenting the aperture region for divergent “wall-eyed” viewing. Orange, Ca2+-bound (i); blue, Ca2+-unbound (j). Green dot, a Cl- ion.

Extended Data Fig. 5 Representative cryo-EM density for bBest2 cryo-EM structures with EGTA only and 5 mM Ca2+.

a–g, Representative map densities for indicated regions are shown with the corresponding atomic model. The Ca2+-unbound state 1 (partially disordered N-terminus, EGTA only), Ca2+-unbound state 2 (completely disordered N-terminus, EGTA only) and Ca2+-bound state (ordered N-terminus, 5 mM Ca2+) structures are shown in green (left), pumpkin (middle) and purple (right), respectively. Green dot, a Ca2+ ion.

Extended Data Fig. 6 Critical residues in Best1 and Best2.

a, b, Population steady-state current density-voltage relationships from HEK293 cells expressing hBest2 S205I (a) or bBest2 G205I (b) in the absence (gray) and presence (black) of 1.2 μM [Ca2+]i; n = 9–20 for each point. c, Bar chart showing the steady-state current densities from HEK293 cells expressing the indicated Best1 and Best2 channels in the absence (gray) and presence (black) of 1.2 μM [Ca2+]i, n = 5–20 for each bar. *P < 0.05 compared to currents without Ca2+ under the same condition, using two-tailed unpaired Student t test. d, e, Population steady-state current density-voltage relationships from HEK293 cells expressing WT bBest2 in the presence of 1.2 μM [Ca2+]i with 120 mM NaCl in the internal solution, and 30 mM NaCl (d) or 120 mM NaSCN (e) in the external solution; n = 5 for each point. f, Population steady-state current density-voltage relationships from HEK293 cells expressing WT bBest2 in the presence of 1.2 μM [Ca2+]i with 120 and 30 mM NaCl in the internal and external solutions, respectively. Results from solutions buffered with NMDG (open circle) were compared to those from solutions buffered with NaOH (solid circle, the same data set as in d); n = 5–6 for each point. g, Population steady-state current density-voltage relationships from HEK293 cells expressing WT bBest2 in the presence of 1.2 μM [Ca2+]i with 120 mM NMDG-Cl and NMDG-CH3SO3 in the internal and external solutions, respectively (open circle), compared to results from 120 mM NaCl and NaCH3SO3 in the internal and external solutions, respectively (solid circle); n = 5–6 for each point. h, Reversal potentials from HEK293 cells expressing bBest2 WT, 3A, H91A, G199A, K208A and K265A. n = 5–18 for each point, the same color labels as in Fig. 3d. The graphs are plotted using the same set of data as in Figs. 3 and 7. All error bars in this figure represent s.e.m.

Extended Data Fig. 7 Expression and membrane trafficking of bBest2 mutants.

a, Top, Western blot showing the transient expression levels of bBest2 WT and mutants in HEK293 whole-cell lysate; Bottom, β-Actin loading control from the same gel. b, Top, cell surface expression levels of bBest2 WT and mutants in HEK293 cells were detected by immunoblotting. Membrane extractions were generated from the same batch of cell pellets as in a; Bottom, quantitation of the levels of bBest2 in plasma membrane from three independent experiments. Data were normalized to the global bBest2 level and then compared to WT. All error bars in this figure represent s.e.m.

Extended Data Fig. 8 A two-gate activation model of bBest2.

a–c, Cartoon showing the effect of Ca2+ on the permeation of Cl- (a), CH3SO3− (b) and I− (c). The number indicates the radius (Å) of the dehydrated anion. The thickness of the arrows reflect conductance of the channel.

Extended Data Fig. 9 A putative anion binding site within the aperture of bBest2.

Left, the molecular model of bBest2. Right, representative map densities with the corresponding atomic model for EGTA Ca2+-unbound state 1 (green), EGTA Ca2+-unbound state 2 (pumpkin), and 5 mM Ca2+-bound state (purple). a, Map processed with C5 symmetry, view from inside the channel towards the aperture, as depicted in the ribbon cartoon (left). b, Map processed with C5 symmetry, view from the side of the aperture, sliced to exclude one protomer, as depicted in the ribbon cartoon (left). c, Map processed with C1 symmetry using the same particle set as its respective C5 map above, same view as in a.

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Owji, A.P., Zhao, Q., Ji, C. et al. Structural and functional characterization of the bestrophin-2 anion channel. Nat Struct Mol Biol 27, 382–391 (2020). https://doi.org/10.1038/s41594-020-0402-z

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