Fig 2: Impact of BEST1 pathogenic variants in photoreceptors. (A) Cryosections obtained from the BD donors and a 65-year-old control were labeled with antibodies specific to rhodopsin (green), while cell nuclei were labeled with TO-PRO-3 (blue). (B) Cryosections were also labeled with antibodies specific to red/green cone opsin (green) and GFAP (red), while cell nuclei have been labeled with TO-PRO-3 (blue). Arrow = abnormal distribution of rhodopsin into cell body; arrowheads = abnormal distribution of red/green cone opsin into the cell body. Bruch’s membrane is indicated by the hashed white line. Scale bar = 40 μm (all images).

Fig 3: 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 ß-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.

Fig 4: Knockdown and rescue of BEST1 gain-of-function mutations in hPSC-RPE cells.(a) Population steady-state current density-voltage relationships in WT hPSC-RPE cells treated with BVSi-Ctrl (black) compared to those in BVSi 3–8 (red) or BVSi 5–4 (blue) treated cells, at 1.2 µM [Ca2+]i, n = 5–17 for each point. *p<0.05 (8.3 × 10-7 for BVSi 3–8 and 1.6 × 10-6 for BVSi 5–4) compared to BVSi-Ctrl treated cells, using two-tailed unpaired Student’s t test. (b) Population steady-state current density-voltage relationships in WT hPSC-RPE cells treated with BVSi 3–8 plus wobble WT BEST1 (blue) compared to those in untreated cells (black), at 1.2 µM [Ca2+]i, n = 5–6 for each point. (c–d) Population steady-state current density-voltage relationships in BEST1I205T/WT (c) or BEST1Y236C/WT (d) hPSC-RPE cells treated with BVSi 3–8 alone (red), or BVSi 3–8 plus wobble WT BEST1 (blue), at 1.2 µM [Ca2+]i, n = 5–9 for each point. *p<0.05 (3.8 × 10-3 for I205T and 2.7 × 10-4 for Y236C) compared to cells treated with BVSi 3–8 alone, using two-tailed unpaired Student’s t test. (e–f) Steady-state current densities recorded at +100 mV plotted vs. [Ca2+]i in BEST1I205T/WT (e) or BEST1Y236C/WT (f) hPSC-RPE cells treated with BVSi 3–8 plus wobble WT BEST1 (blue) compared to those in untreated WT hPSC-RPE (black), n = 5–6 for each point. The plots were fitted to the Hill equation. All error bars in this figure represent s.e.m.

Fig 5: Impact of BEST1 pathogenic variants in RPE autofluorescent granules. Cryosections obtained from the BD donors and a 95-year-old control were observed using the green channel (FITC filter) and red channel (TRITC filter). Autofluorescence was overlaid on differential interference contrast (DIC) images. Bruch’s membrane is indicated by hashed white line, asterisk = drusen. Scale bar = 40 µm (all images).