1. Structural Biology and Molecular Biophysics
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Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca2+-dependent Cl- currents in human RPE

  1. Yao Li
  2. Yu Zhang
  3. Yu Xu
  4. Alec Kittredge
  5. Nancy Ward
  6. Shoudeng Chen
  7. Stephen H Tsang  Is a corresponding author
  8. Tingting Yang  Is a corresponding author
  1. New York Presbyterian Hospital/Columbia University, United States
  2. School of Medicine and Dentistry, University of Rochester, United States
  3. Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, China
  4. The Fifth Affiliated Hospital of Sun Yat-sen University, China
Research Article
Cite this article as: eLife 2017;6:e29914 doi: 10.7554/eLife.29914
8 figures, 2 tables, 2 data sets and 1 additional file

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 protocol used to elicit currents is shown in Insert. Scale bar, 1 nA, 150 ms. (C) Population steady-state current-voltage relationships at different free [Ca2+]i; n = 5–6 for each point. The plot was fitted to the Hill equation. (D) Ca2+-dependent activation of surface current. Steady-state current density recorded at +100 mV plotted vs. free [Ca2+]i; n = 5–6 for each point. See also Figure 1—figure supplements 1 and Figure 1—source data 1.

https://doi.org/10.7554/eLife.29914.003
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) Confocal images showing plasma membrane localization of BEST1. Scale bar, 10 μm. (D) Comparison of current amplitudes in iPSC-RPEs from two BEST1 WT donors. Bar chart showing the steady-state current amplitudes at 0 [Ca2+]i, 1.2 μM [Ca2+]i, and 1.2 μM [Ca2+]i + 100 μM NFA in RPEs from two distinct BEST1 WT human donors; n = 5–6. ∗$p<0.05 compared to current amplitudes at 1.2 μM [Ca2+]i from donor #1 and #2, respectively, using two-tailed unpaired Student t test.

https://doi.org/10.7554/eLife.29914.004
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 ms. (C) Population steady-state current-voltage relationships at different free [Ca2+]i; n = 5–6 for each point. (D) Ca2+-dependent activation of surface currents in fhRPE () and iPSC-RPE (●). Steady-state current density recorded at +100 mV plotted vs. free [Ca2+]i; n = 5–6 for each point. The plots were fitted to the Hill equation. (E) Bar chart showing the steady-state current amplitudes at 0 and 18 μM free [Ca2+]i in RPEs from two distinct human fetuses; n = 5–6. ∗$p<0.05 compared to fetus #1 (0.02) and #2 (0.02), respectively, at 18 μM [Ca2+]i using two-tailed unpaired Student t test. See also Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.29914.006
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 showing different time-dependent activation under different [Ca2+]i; n = 5–6 for each bar. *p<0.05 compared to 1 min at 18 μM [Ca2+]i using two-tailed unpaired Student t test.

https://doi.org/10.7554/eLife.29914.007
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 vitelliform deposits (red arrow) superior to the optic disks and encircling the maculae. (B) SDOCTs of the macula in patient 1 and patient 2. Scale bar, 200 μm. In Patient 1, there are bilateral, multifocal serous retinal detachments involving the maculae and cystoid deposits in the macula (red arrow). Patient 2 presents a relative preservation of the retina change compared to patient 1. (C) ERGs of patient 1 and patient 2 (red lines), right and left eyes, respectively, show extinguished maximum response amplitudes between a- and b-waves, compared to those from age matched BEST1 WT controls (black lines). See also Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.29914.008
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.

https://doi.org/10.7554/eLife.29914.009
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). (B) Confocal images showing diminished plasma membrane localizations of BEST1 P274R, and normal plasma membrane localization of BEST1 I201T. Scale bar,15 μm. (C) Representative current traces recorded from patient iPSC-RPEs at 1.2 μM [Ca2+]i. Scale bar, 500 pA, 150 ms. (D) Population steady-state current-voltage relationships in BEST1 WT (●), P274R () and I201T () iPSC-RPEs at 1.2 μM [Ca2+]i; n = 5–6 for each point. p<0.05 (2 × 10−7 for P274R and 6 × 10−4 for I201T) compared to WT using two-tailed unpaired Student t test. Insert, confocal images showing P274R iPSC-RPE in bright field. Scale bar,10 μm. (E) CaCC currents in BEST1 P274R patient iPSC-RPE were rescued by complementation with WT BEST1-GFP. Complementation (, n = 5–6 for each point), compared to BEST1 P274R (, n = 3–5 for each point), and WT (●). The plots were fitted to the Hill equation. Insert, confocal images showing P274R iPSC-RPE complemented with WT BEST1-GFP expressed from a BacMam baculoviral vector. Scale bar,10 μm. (F) Ca2+-dependent currents in BEST1 I201T iPSC-RPE () compared to WT iPSC-RPE (●). Steady-state current density recorded at +100 mV plotted vs. free [Ca2+]i; n = 5–6 for each point. The plots were fitted to the Hill equation. See also Figure 4—figure supplement 1 and Figure 1—source data 1.

https://doi.org/10.7554/eLife.29914.010
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. Scale bar, 1 nA, 100 ms. (B) Population steady-state current-voltage relationships in BEST1-GFP complementation (), compared to BEST1 P274R () and WT (●); n = 5–6 for each point. #p<0.05 compared to WT (2 × 10−7) or complementation (0.01) using one-way ANOVA and Bonferroni post hoc analyses. (C) Bar chart showing the steady-state current amplitudes at 1.2 μM [Ca2+]i and 1.2 μM [Ca2+]i +100μM NFA in P274R patient iPSC-RPE over-expressing BEST1-GFP; n = 5–6. p<0.05 compared to current amplitudes at 1.2 μM [Ca2+]i, using two-tailed unpaired Student t test. (D) Normalized Ca2+-dependent currents in BEST1 I201T iPSC-RPE () compared to WT iPSC-RPE (●). The plots were fitted to the Hill equation.

https://doi.org/10.7554/eLife.29914.011
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 (●), P274R () and I201T () at 1.2 μM [Ca2+]i; n = 5–6 for each point. ∗#p<0.05 compared to WT (8 × 10−4 for P274R and 0.01 for I201T) or to I201T (0.04), respectively, using one-way ANOVA and Bonferroni post hoc analyses. See also Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.29914.012
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 = 5–6. p<0.05 compared to current amplitudes at 1.2 μM [Ca2+]i in HEK293 cells transfected with BEST1 WT using two-tailed unpaired Student t test. (C) Western blot showing similar expression levels of transiently transfected BEST1 WT, P274R and I201T in HEK293 cells.

https://doi.org/10.7554/eLife.29914.013
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 are shown with the extracellular side on the top. The side chains of critical residues are in red. (B) Location of the patient mutations in relationship to the channel pore. Left, as viewed from the side; right, from inside the plasma membrane. (C) Visualization of the location of I201T. The side chains of critical residues are in red. See also Figure 6—figure supplements 1 and 2.

https://doi.org/10.7554/eLife.29914.014
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) Critical residues in hBest1 (BEST1), mBest2 (mouse bestrohpin2), dBest1 (Drosophila melanogaster bestrophin1) and cBest1 (chicken bestrophin1). Numbers showing the positions of residues in hBest1. (D) Visualization of P274 and the predicted steric clash by the P274R mutation. The side chains of critical residues are in red.

https://doi.org/10.7554/eLife.29914.015
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 sequences. The secondary structures of KpBest and cBest1 are labeled above and underneath the sequences, respectively.

https://doi.org/10.7554/eLife.29914.016
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 test. (B) Current trace of KpBest WT and mutant single channels recorded from planar lipid bilayers at 80 mV with 150 mM NaCl in both cis and trans solutions. Scale bar, 2.5 pA, 250 ms. (C) Histograms showing single channel current amplitudes of KpBest WT and the L177T mutant. n = 3.

https://doi.org/10.7554/eLife.29914.017
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.

https://doi.org/10.7554/eLife.29914.018
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.

https://doi.org/10.7554/eLife.29914.019

Tables

Table 1
Summary of disease-causing mechanisms of BEST1 P274R and I201T mutations.
https://doi.org/10.7554/eLife.29914.021
MechanismSystemP274RI201T
Phenotype-PatientSevereMild
FunctionICaC currentRPENullSmall
Ca2+ sensitivityRPEN/ANormal
CaC current of BEST1HEK293NullSmall
NBEST1 expressionRPENormalNormal
Membrane localizationRPEDiminishedNormal
iUnitary currentKpBestNullSmall
Structure-KpBest crystal + human modelDisruptedSlightly altered
  1. 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
DesignationSource or referenceIdentifiersAdditional information
gene (human)BEST1PMID: 25324390
gene
(Klebsiella pneumoniae)
KpBestPMID: 25324390
strain, strain
background (E.coli)
DH5alphaotherLaboratory of
Wayne Hendrickson
strain, strain
background (E. coli)
BL21 plysSotherLaboratory of
Wayne Hendrickson
cell line (human)HEK293otherRRID:CVCL_0045Laboratory of
David Yule
transfected
construct (human)
pEGFP-N1-BEST1 WTPMID: 25324390
transfected
construct (human)
pEGFP-N1-BEST1 I201Tthis paperMade from
pEGFP-N1-BEST1
WT by site-directed mutagenesis
transfected
construct (human)
pEGFP-N1-BEST1 P274Rthis paperMade from
pEGFP-N1-BEST1 WT by
site-directed mutagenesis
biological
sample (human)
skin cellsotherNew York
Presbyterian Hospital
biological
sample (human)
fetus eye samplesotherNew York
Presbyterian Hospital
biological
sample (human)
BEST1 WT iPSC-RPEthis paperGenerated from
donor skin cells by
re-programming and
differentiation
biological
sample (human)
BEST1 I201T iPSC-RPEthis paperGenerated from
donor skin cells by
re-programming and
differentiation
biological
sample (human)
BEST1 P274R iPSC-RPEthis paperGenerated from
donor skin cells by
re-programming and
differentiation
antibodyBESTROPHIN1Novus Biologicals NB300-164RRID:AB_100030191:200
antibodyZO-1Invitrogen 40–2200RRID:AB_25334561:500
antibodyAlexa Fluor 488-conjugated IgGInvitrogen A-11070RRID:AB_25341141:1000
antibodyAlexa Fluor 555-conjugated IgGInvitrogen A-21422RRID:AB_25358441:1000
antibodyRPE65Novus Biologicals NB100-355RRID:AB_100021481:1000
antibodyCRALBPAbcam ab15051RRID:AB_22694741:500
antibodyβ-actinAbcam ab8227RRID:AB_23051861:2000
antibodyGFPInvitrogen A6455RRID:AB_2215701:5000
antibodySOX2, Tra-1–60, SSEA4, NanogAbcam ab1098841:200
antibodyEEA1Fisher Scientific MA5-14794RRID:AB_109858241:200
recombinant
DNA reagent
pEG BacmamotherLaboratory of Eric Gouaux
recombinant
DNA reagent
pEG Bacmam-BEST1-GFPthis paperMade from
pEG Bacmam by
inserting BEST1-GFP
recombinant
DNA reagent
BEST1-GFP Bacmam virusthis paperProduced from
pEG Bacmam-BEST1-GFP
by published protocols
(Goehring et al., 2014)
recombinant
DNA reagent
pMCSG7-10xHis-KpBestΔC11PMID: 25324390
recombinant
DNA reagent
pMCSG7-10xHis-KpBestΔC11
L177T
this paperMade from
pMCSG7-
10xHis-KpBestΔC11
by site-directed
mutagenesis
recombinant
DNA reagent
pMCSG7-10xHis-KpBestΔC11
P239R
this paperMade from
pMCSG7-
10xHis-KpBestΔC11
by site-directed
mutagenesis
sequence-based
reagent
BEST1 I201T forward primerthis paperACCCGGGACC
CTATCCTGCT
sequence-based
reagent
BEST1 I201T reverse primerthis paperGATAGGGTCCCGGG
TTCGACCTCCAAGCCACG
sequence-based
reagent
BEST1 P274R forward primerthis paperCGCGTCTTCAC
GTTCCTGCAGTT
sequence-based
reagent
BEST1 P274R reverse primerthis paperGAACGTGAAGAC
GCGCACAACGAGGT
sequence-based
reagent
KpBest L177T forward primerthis paperACCAGCGACA
TCACTTACGGGC
sequence-based
reagent
KpBest L177T reverse primerthis paperAGTGATGTCGCT
GGTCTTGCCCGCCTCCCG
sequence-based
reagent
KpBest P239R forward primerthis paperCGGTTTGTCTCGGTC
TTTATCTCTTACACC
sequence-based
reagent
KpBest P239R reverse primerthis paperGACCGAGAC
AAACCGCGTCA
TGTA GTGCAGATCGC
peptide,
recombinant protein
KpBestΔC11 L177Tthis paperExpressed from
E. coli BL21 plysS,
and purified by
affinity and
size-exclusion
chromatography
peptide,
recombinant protein
KpBestΔC11 P239Rthis paperExpressed 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 KitClontech 639645
chemical compound,
drug
mTeSR-1 mediumSTEMCELL Technologies 5850
chemical compound,
drug
matrigelCORNING 356230
chemical compound,
drug
nicotinamideSigma-Aldrich N0636
chemical compound,
drug
Activin-APeproTech 120–14
software, algorithmXDSPMID: 20124692
software, algorithmPhaserPMID: 19461840RRID:SCR_014219
software, algorithmPhenixPMID: 20124702RRID:SCR_014224
software, algorithmCootPMID: 15572765RRID:SCR_014222
software, algorithmPyMOLhttp://www.pymol.org/RRID:SCR_000305
software, algorithmOriginhttp://www.originlab.com/index.aspx?go=PRODUCTS/OriginRRID:SCR_014212
software, algorithmMODELLERPMID: 14696385RRID:SCR_008395

Data availability

The following data sets were generated
  1. 1
The following previously published data sets were used
  1. 1
    Crystal structure of a bacterial Bestrophin homolog from Klebsiella pneumoniae
    1. Yang T
    2. Liu Q
    3. Hendrickson WA
    (2014)
    Publicly available at the RCSB PDB website (accession no: 4WD8).

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