Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca2+-dependent Cl- currents in human RPE
- New York Presbyterian Hospital/Columbia University, United States
- School of Medicine and Dentistry, University of Rochester, United States
- Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, China
- The Fifth Affiliated Hospital of Sun Yat-sen University, China
Figures
Figure 1 with 1 supplement
Subcellular localization of BEST1 and surface Ca2+-dependent Cl- current in BEST1 WT donor iPSC-RPEs.
(A) Confocal images showing plasma membrane localization of BEST1. Scale bar, 10 μm. (B) Representative current traces recorded from a BEST1 WT donor iPSC-RPEs at various free [Ca2+]i. Voltage …
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Figure 1—source data 1
Comparison of different data sets from the same donors.
Ca2+-dependent Cl- current amplitudes in two clonal iPSC-RPEs (for WT and I201T) or iPSC-RPEs generated by two different sets of differentiations (for P274R) from the same donors. n = 5–6 for each data set. Diff: differentiation.
- https://doi.org/10.7554/eLife.29914.005
Figure 1—figure supplement 1
Characterization of WT iPSC and iPSC-RPE.
(A) Phase picture of established WT iPSC line before differentiation. Scale bar, 400 μm. (B) Immunocytofluorescence images of pluripotency markers in established iPSC. Scale bar, 200 μm. (C) …
Figure 2 with 1 supplement
Subcellular localization of BEST1 and surface Ca2+-dependent Cl- current in fhRPEs.
(A) Confocal images showing plasma membrane localization of BEST1. Scale bar, 10 μm. (B) Representative current traces recorded from a BEST1 WT fhRPEs at various free [Ca2+]i. Scale bar, 1 nA, 150 …
Figure 2—figure supplement 1
The Ca2+and time-dependent activation of surface Cl- current in fhRPE.
(A) Representative current traces recorded from fhRPEs at 18 μM [Ca2+]i. Scale bar, 1 nA, 150 ms. (B) Time-dependent activation of surface Cl- current amplitudes when [Ca2+]i is 18 μM. (C) Bar chart …
Figure 3 with 1 supplement
Clinical phenotypes of two patients with BEST1 mutations.
(A) Color fundus photographs from patient 1 (P274R) and patient 2 (I201T), right and left eyes, respectively. Both of the patients’ fundus show bilateral, confluent curvilinear subretinal yellowish …
Figure 3—figure supplement 1
Reduced EOG light peak in patient with BEST1 I201T mutation.
The EOG of BEST1 I201T patient (red) was compared to that of a similarly aged BEST1 WT person (black). Scale bar, 2 μV/deg, 5 min.
Figure 4 with 1 supplement
Subcellular localization of BEST1 and surface Ca2+-dependent Cl- current in patient-derived iPSC-RPEs.
(A) Western blots show similar BEST1 expression levels in WT and patient-derived iPSC-RPEs. Each sample was from one cell lysis (BEST1 and β-actin, RPE65 and CRALBP were on two gels, respectively). …
Figure 4—figure supplement 1
CaCC currents in BEST1 patient iPSC-RPEs.
(A–C) P274R patient iPSC-RPE were rescued by complementation with WT BEST1-GFP at 1.2 μM [Ca2+]i. (A) Representative current traces recorded from P274R patient iPSC-RPE over-expressing WT BEST1-GFP. …
Figure 5 with 1 supplement
Surface Ca2+-dependent Cl- current in HEK293 cells expressing WT and mutant BEST1.
(A) Representative current traces recorded from transfected HEK293 cells at 1.2 μM [Ca2+]i. Scale bar, 150 pA, 150 ms. (B) Population steady-state current-voltage relationships for BEST1 WT (●), …
Figure 5—figure supplement 1
Ca2+-dependent Cl- current in BEST1 transfected HEK293 cells.
(A) Representative current traces recorded from untransfected HEK293 cells at 1.2 μM [Ca2+]i. Scale bar, 150 pA, 150 ms. (B) Bar chart showing population steady-state current amplitudes at 100 mV; n …
Figure 6 with 2 supplements
Patient mutations in a BEST1 homology model.
(A) Left, ribbon diagram of the BEST1 pentamer with each protomer colored differently, as viewed from the side. Right, ribbon diagram of two oppositely facing (144°) protomers of a BEST1 pentamer …
Figure 6—figure supplement 1
Structural analysis of BEST1 mutations in a homology model.
(A) 2D topology of a human BEST1 protomer, colored spectrally from blue at its N-terminal segment to red at its C-terminal segment. (B) Ribbon diagram of a human BEST1 protomer. Colored as in A. (C) …
Figure 6—figure supplement 2
Structure-based sequence alignment of KpBest, hBest1 and cBest1.
The KpBest structure has been used to restrict sequence gaps to inter-helical segments. Black background, identical residues in all three sequences; grey background, identical residues in two …
Figure 7
Influence of patient mutations on single channel conductance.
(A) Bar chart showing purified KpBest WT and mutant pentameric protein per wet cell yields. n = 3 for each bar. ∗p<0.05 compared to WT (2 × 10−3) or L177T (0.03) using two-tailed unpaired Student t …
Figure 8 with 1 supplement
Superposition of KpBest WT with L177T mutant based on regional alignment of residues 174–180.
Ribbon diagram of the KpBest WT chain A (blue) and KpBest L177T chain A (green) with highlighted stick diagram of residue 177. See also Figure 8—figure supplement 1 and Figure 8—source data 1.
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Figure 8—source data 1
Data collection and refinement statistics of KpBest L177T.
aStatistics for the highest-resolution shell are shown in parentheses.
- https://doi.org/10.7554/eLife.29914.020
Figure 8—figure supplement 1
Crystal structure of KpBest L177T.
Stereo images of the electron density maps (2Fo-Fc map, 1.2σ level) of KpBest WT (top, 2.3 Å) and L177T (bottom, 3.1 Å) presenting residues 174–180 for divergent ‘wall-eyed’ viewing.
Tables
Table 1
Summary of disease-causing mechanisms of BEST1 P274R and I201T mutations.
https://doi.org/10.7554/eLife.29914.021| Mechanism | System | P274R | I201T | ||
|---|---|---|---|---|---|
| Phenotype | - | Patient | Severe | Mild | |
| Function | I | CaC current | RPE | Null | Small |
| Ca2+ sensitivity | RPE | N/A | Normal | ||
| CaC current of BEST1 | HEK293 | Null | Small | ||
| N | BEST1 expression | RPE | Normal | Normal | |
| Membrane localization | RPE | Diminished | Normal | ||
| i | Unitary current | KpBest | Null | Small | |
| Structure | - | KpBest crystal + human model | Disrupted | Slightly altered | |
-
I = N × Po× i. I, whole-cell current amplitude; N, number of surface channels; Po, channel open probability; i, unitary current.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (human) | BEST1 | PMID: 25324390 | ||
| gene (Klebsiella pneumoniae) | KpBest | PMID: 25324390 | ||
| strain, strain background (E.coli) | DH5alpha | other | Laboratory of Wayne Hendrickson | |
| strain, strain background (E. coli) | BL21 plysS | other | Laboratory of Wayne Hendrickson | |
| cell line (human) | HEK293 | other | RRID:CVCL_0045 | Laboratory of David Yule |
| transfected construct (human) | pEGFP-N1-BEST1 WT | PMID: 25324390 | ||
| transfected construct (human) | pEGFP-N1-BEST1 I201T | this paper | Made from pEGFP-N1-BEST1 WT by site-directed mutagenesis | |
| transfected construct (human) | pEGFP-N1-BEST1 P274R | this paper | Made from pEGFP-N1-BEST1 WT by site-directed mutagenesis | |
| biological sample (human) | skin cells | other | New York Presbyterian Hospital | |
| biological sample (human) | fetus eye samples | other | New York Presbyterian Hospital | |
| biological sample (human) | BEST1 WT iPSC-RPE | this paper | Generated from donor skin cells by re-programming and differentiation | |
| biological sample (human) | BEST1 I201T iPSC-RPE | this paper | Generated from donor skin cells by re-programming and differentiation | |
| biological sample (human) | BEST1 P274R iPSC-RPE | this paper | Generated from donor skin cells by re-programming and differentiation | |
| antibody | BESTROPHIN1 | Novus Biologicals NB300-164 | RRID:AB_10003019 | 1:200 |
| antibody | ZO-1 | Invitrogen 40–2200 | RRID:AB_2533456 | 1:500 |
| antibody | Alexa Fluor 488-conjugated IgG | Invitrogen A-11070 | RRID:AB_2534114 | 1:1000 |
| antibody | Alexa Fluor 555-conjugated IgG | Invitrogen A-21422 | RRID:AB_2535844 | 1:1000 |
| antibody | RPE65 | Novus Biologicals NB100-355 | RRID:AB_10002148 | 1:1000 |
| antibody | CRALBP | Abcam ab15051 | RRID:AB_2269474 | 1:500 |
| antibody | β-actin | Abcam ab8227 | RRID:AB_2305186 | 1:2000 |
| antibody | GFP | Invitrogen A6455 | RRID:AB_221570 | 1:5000 |
| antibody | SOX2, Tra-1–60, SSEA4, Nanog | Abcam ab109884 | 1:200 | |
| antibody | EEA1 | Fisher Scientific MA5-14794 | RRID:AB_10985824 | 1:200 |
| recombinant DNA reagent | pEG Bacmam | other | Laboratory of Eric Gouaux | |
| recombinant DNA reagent | pEG Bacmam-BEST1-GFP | this paper | Made from pEG Bacmam by inserting BEST1-GFP | |
| recombinant DNA reagent | BEST1-GFP Bacmam virus | this paper | Produced from pEG Bacmam-BEST1-GFP by published protocols (Goehring et al., 2014) | |
| recombinant DNA reagent | pMCSG7-10xHis-KpBestΔC11 | PMID: 25324390 | ||
| recombinant DNA reagent | pMCSG7-10xHis-KpBestΔC11 L177T | this paper | Made from pMCSG7- 10xHis-KpBestΔC11 by site-directed mutagenesis | |
| recombinant DNA reagent | pMCSG7-10xHis-KpBestΔC11 P239R | this paper | Made from pMCSG7- 10xHis-KpBestΔC11 by site-directed mutagenesis | |
| sequence-based reagent | BEST1 I201T forward primer | this paper | ACCCGGGACC CTATCCTGCT | |
| sequence-based reagent | BEST1 I201T reverse primer | this paper | GATAGGGTCCCGGG TTCGACCTCCAAGCCACG | |
| sequence-based reagent | BEST1 P274R forward primer | this paper | CGCGTCTTCAC GTTCCTGCAGTT | |
| sequence-based reagent | BEST1 P274R reverse primer | this paper | GAACGTGAAGAC GCGCACAACGAGGT | |
| sequence-based reagent | KpBest L177T forward primer | this paper | ACCAGCGACA TCACTTACGGGC | |
| sequence-based reagent | KpBest L177T reverse primer | this paper | AGTGATGTCGCT GGTCTTGCCCGCCTCCCG | |
| sequence-based reagent | KpBest P239R forward primer | this paper | CGGTTTGTCTCGGTC TTTATCTCTTACACC | |
| sequence-based reagent | KpBest P239R reverse primer | this paper | GACCGAGAC AAACCGCGTCA TGTA GTGCAGATCGC | |
| peptide, recombinant protein | KpBestΔC11 L177T | this paper | Expressed from E. coli BL21 plysS, and purified by affinity and size-exclusion chromatography | |
| peptide, recombinant protein | KpBestΔC11 P239R | this paper | Expressed from E. coli BL21 plysS, and purified by affinity and size-exclusion chromatography | |
| commercial assay or kit | CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo Fisher Scientific A16517 | ||
| commercial assay or kit | In-fusion Cloning Kit | Clontech 639645 | ||
| chemical compound, drug | mTeSR-1 medium | STEMCELL Technologies 5850 | ||
| chemical compound, drug | matrigel | CORNING 356230 | ||
| chemical compound, drug | nicotinamide | Sigma-Aldrich N0636 | ||
| chemical compound, drug | Activin-A | PeproTech 120–14 | ||
| software, algorithm | XDS | PMID: 20124692 | ||
| software, algorithm | Phaser | PMID: 19461840 | RRID:SCR_014219 | |
| software, algorithm | Phenix | PMID: 20124702 | RRID:SCR_014224 | |
| software, algorithm | Coot | PMID: 15572765 | RRID:SCR_014222 | |
| software, algorithm | PyMOL | http://www.pymol.org/ | RRID:SCR_000305 | |
| software, algorithm | Origin | http://www.originlab.com/index.aspx?go=PRODUCTS/Origin | RRID:SCR_014212 | |
| software, algorithm | MODELLER | PMID: 14696385 | RRID:SCR_008395 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29914.022
