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.2012 Jan 15;441(2):685-96.
doi: 10.1042/BJ20111004.

Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death

Affiliations

Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death

Deborah L Duricka et al. Biochem J..

Abstract

Mutations that perturb the function of photoreceptor CNG (cyclic nucleotide-gated) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the ER (endoplasmic reticulum) is known to cause ER stress and trigger the UPR (unfolded protein response), an evolutionarily conserved cellular programme that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared with wild-type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones {TUDCA (tauroursodeoxycholate sodium salt), 4-PBA (sodium 4-phenylbutyrate) and the cGMP analogue CPT-cGMP [8-(4-chlorophenylthio)-cGMP]} differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER-stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization-defective CNG channels, and may represent a contributing factor for photoreceptor degeneration.

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Figures

Figure 1
Figure 1. Disease-associated mutations R563H and Q655X impair plasma-membrane localization and increase ER retention of CNGA3 channel subunits
(A) CNGA3 subunit topology and location of mutations.(B–G) Expression and localization of human cone CNGA3 and CNGB3 subunits in 661W cells, determined using cell-surface biotinylation with avidin-retrieval(B, C) or confocal microscopy(D–G).(B) Representative immunoblot showing total (1% of input, with actin for normalization) and surface localized CNGA3 subunits.(C) Cell-surface localization of CNGA3 subunits, displayed as avidin pullouts from (B) relative to wild type, is impaired by R563H and Q655X mutations. Data are means ± SEM of six independent experiments; *p < 0.05.(D) Confocal fluorescence microscopy images and graphs showing plasma membrane (PM) marked with wheat germ agglutinin (red) and FLAG-CNGA3 (green); PM localization (yellow) of R563H and Q655X subunits is impaired.(E) Colocalization calculated for regions encompassing cross-sections in (D) using Pearson’s product-moment correlation coefficient, showing decreased PM localization of R563H and Q655X subunits. Images are 0.4–0.6 micron optical Z sections, representative of > 10 cells each; data are means ± SEM (n = 6 cells); *p < 0.0001.(F) Confocal images showing CNGA3 subunits (green) and endoplasmic reticulum (ER) marked with DsRed2-ER (blue).(G) Colocalization calculated for regions defined in (F) using Pearson’s product-moment correlation coefficient, showing increased ER localization of R563H and Q655X subunits. Images are 0.4–0.6 micron optical Z sections, representative of > 10 cells each; data are means ± SEM (n = 6 cells); *p < 0.05.
Figure 2
Figure 2. Localization-defective channels display altered stability and protease sensitivity
(A–D) Amount of CNGA3 and CNGB3 subunits remaining at indicated time points after arrest of protein synthesis.(A, B) Representative immunoblots of CNGA3 or CNGB3 subunits, respectively.(C) Time-dependent decrease of CNGA3 subunits expressed as percent of initial amount, showing increased degradation rate for Q655X relative to wild type.(D) Percent initial amount of CNGB3, with indicated CNGA3 subunits, showing that trafficking defective CNGA3 subunits increased the degradation rate of coexpressed CNGB3 subunits. Data are means ± SEM of seven independent experiments. *p < 0.05.(E, F) Protease-sensitivity assay to probe CNGA3 subunit folding.(E) Representative western blot for proteolysis of purified channels.(F) Percent initial amount of CNGA3 remaining after trypsin proteolysis at indicated time points, showing altered trypsin sensitivity. Data are means ± SEM of three independent experiments; *p < 0.05.
Figure 3
Figure 3. Pharmacological stress induces the unfolded protein response in 661W cells
(A–D) RT-qPCR quantitation of XBP1 (spliced and total), BiP, CHOP, and HPRT (internal standard) mRNA from cells treated with DTT, thapsigargin (Tg), or tunicamycin (Tn), relative to untreated cells. Percentage of XBP1 spliced(A) determined by percentage of spliced:total mRNA. Fold changes of total XBP1(B), BiP(C), and CHOP(D).(E, F) Immunoblot quantitation of phosphorylated PERK (P-PERK), BiP, CHOP, and actin after treatment with Tg or Tn.(E) Representative immunoblot.(F) Levels of P-PERK, BiP, and CHOP (relative to actin) after treatments indicated in (E) normalized to levels in untreated cells. Data are means ± SEM of 3–6 independent experiments; *p < 0.05.
Figure 4
Figure 4. Localization defective CNG channels selectively induce the unfolded protein response
RT-qPCR and immunoblot quantitation of ER stress markers in cells expressing CNGA3 and CNGB3 subunits.(A) Percent of XBP1 spliced and fold changes of BiP and CHOP mRNA were normalized to values in cells expressing wild-type channels. Data are means ± SEM of three independent experiments; *p < 0.05.(B) Representative immunoblot showing expression of P-PERK, BiP, CHOP, and actin.(C) Amounts of P-PERK, BiP, and CHOP (relative to actin) normalized to levels in cells expressing wild-type channels. Data are means ± SEM of 4–6 independent experiments; *p < 0.05.
Figure 5
Figure 5. Chemical and pharmacological chaperones reduce mRNA indicators of ER stress induced by localization defective CNG channels
RT-qPCR analysis of chaperone-treated cells expressing CNGA3 and CNGB3 subunits. Percent of XBP1 spliced(A) and fold changes of BiP(B) and CHOP(C) mRNA normalized to values in untreated cells expressing wild-type channels. Data are means ± SEM of 4–5 independent experiments; #p < 0.05 compared to untreated cells expressing wild-type channels; *p < 0.05 compared to untreated cells.
Figure 6
Figure 6. Chaperones selectively reduce BiP and CHOP protein levels induced by localization defective CNG channels
Immunoblot analysis of chaperone-treated cells expressing CNGA3 and CNGB3 subunits.(A) Representative immunoblot. Amounts of BiP(B) and CHOP(C) protein (relative to actin) normalized to levels in untreated cells expressing wild-type channels. Data are means ± SEM of 4–5 independent experiments. #p < 0.05 compared to untreated cells expressing wild-type channels; *p < 0.05 compared to untreated cells.
Figure 7
Figure 7. Chaperones selectively enhance stability of localization defective CNGA3-Q655X subunits
Immunoblot analysis of proteins in chaperone-treated cells expressing CNGA3 and CNGB3 subunits after cycloheximide protein synthesis arrest.(A) Representative immunoblot.(B) Amount of CNGA3 subunit protein remaining after 4 hours relative to initial amount, normalized to values in untreated cells. Data are means ± SEM of four independent experiments; *p < 0.05 compared to untreated cells.
Figure 8
Figure 8. Chaperones selectively enhance cell-surface localization of CNGA3-R563H subunits
Immunoblot analysis of CNGA3 plasma membrane (PM) localization using cell-surface biotinylation with avidin-retrieval.(A) Representative immunoblot showing total (1% of input) and surface localized CNGA3 subunits.(B) PM localization of CNGA3 subunits, displayed relative to untreated cells expressing wild-type channels. Data are means ± SEM of three independent experiments; #p < 0.05 compared to untreated cells expressing wild-type channels; *p < 0.05 compared to untreated cells.
Figure 9
Figure 9. Chaperones selectively enhance plasma-membrane localization and reduce ER retention of localization defective CNGA3-R563H and -Q655X subunits
Colocalization of CNGA3 (green) with PM (red) or ER (blue), determined by confocal fluorescence microscopy.(A, C) Representative confocal images.(B, D) Colocalization calculated for regions defined in (A) or (C) using Pearson’s product-moment correlation coefficient, normalized to untreated cells expressing wild-type channels. Images are 0.4–0.6 micron optical Z sections, representative of > 10 cells each; data are means ± SEM (n = 4 cells); #p < 0.05 compared to untreated cells expressing wild-type channels; *p < 0.05 compared to untreated cells.
Figure 10
Figure 10. Chaperones improve survival of cells expressing localization defective CNG channels
Quantitation of relative cell death, using lactate dehydrogenase (LDH) release assay. Percent cell death (relative to vector-only transfected cells), normalized to untreated cells expressing wild-type channels, showing rescue of cell viability with chaperone treatment. Data are means ± SEM of 3–5 independent experiments; #p < 0.02 compared to untreated cells expressing wild-type channels; *p < 0.01 compared to untreated cells.
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