XB-ART-47275Mol Vis 2013 Jun 11;19:1268-81. doi: 10.1167/13.9.1268.
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Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells.
PURPOSE: To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. METHODS: Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3'',5''-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3'',5''-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. RESULTS: Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. CONCLUSIONS: These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.
PubMed ID: 23805033
PMC ID: PMC3692405
Article link: Mol Vis
Species referenced: Xenopus
Genes referenced: actb actl6a adm cnga3 cngb3
GO keywords: cyclic nucleotide-gated ion channel activity
Disease Ontology terms: achromatopsia 3
OMIMs: ACHROMATOPSIA 3; ACHM3
Article Images: [+] show captions
|Figure 1. Disease-associated mutations in CNGB3 alter the gating properties of heteromeric channels. A: Representative current traces are shown for CNGA3 plus CNGB3 channels after activation by saturating concentrations of CPT-cGMP (4 μM) or CPT-cAMP (100 μM). Current traces were elicited by voltage steps from a holding potential of 0 mV to +80 mV, −80 mV, and then back to 0 mV. B: Representative dose–response relationships for CPT-cGMP activation of CNG channels, after expression of CNGA3 plus CNGB3-WT (circles), T383fsX (squares), or F525N (triangles) subunits. Currents were normalized to the maximum cGMP current. Continuous curves represent fits of the dose–response relationship with the Hill equation as described in the Methods section. The parameters for each channel type were as follows: for WT, K1/2,CPT-cGMP=248 nM, h=1.5; for T383fsX, K1/2, cGMP=111 nM, h=1.9; and for F525N, K1/2,CPT-cGMP=79 nM, h=1.7.|
|Figure 2. Disease-associated mutations in CNGB3 increase cytotoxicity. A: Western blot demonstrating expression of FLAG-tagged wild-type (WT) and mutant cone CNG channel subunits in 661W cells following transfection with indicated plasmids (above). Approximate locations of molecular weight markers (in kilodaltons) are indicated to the right of the immunoblot. Cell lysates were also probed with beta-actin antibody (below). The molecular weight of beta actin was ~42 kDa. B: The bar graph demonstrates increased cytotoxicity (measured as LDH release from dying cells) for cells expressing CNGA3 plus wild-type (WT) or mutant CNGB3 exposed to various concentrations of CPT-cGMP for 24 h (n=46 to 48). Cytotoxicity was normalized to that of control cells transfected with vector (pOPRSVI) alone, incubated in the absence of CPT-cGMP; *, significant difference between groups indicated by bracket (p<0.05); +, significant difference between F525N groups with or without 0.1 μM CPT-cGMP treatment (p<0.01).|
|Figure 3. CNGB3 F525N mutation impairs cell viability with CPT-cGMP treatment. Transfected 661W cells were treated with or without 0.1 μM CPT-cGMP for 24 h. After treatment, cells were labeled according to the LIVE/DEAD assay protocol: vital cells were stained by calcein AM and show green fluorescence (A, C, E, and G); damaged cells were penetrated by ethidium homodimer and show red fluorescent nuclei (B, D, F and H). Fluorescent images were obtained using a Zeiss LSM 510 confocal laser-scanning microscope as described in the Methods section.|
|Figure 4. CNGB3 F525N mutation increases annexin V–positive cells compared to wild-type channels. Transfected cells were treated with 0.1 μM CPT-cGMP for 24 h. For determination of cell death, cells were stained with fluorescein-labeled annexin V, a protein with a high affinity for phosphatidylserine (A and B). Cells were also counterstained with DAPI to count nuclei (C and D), and the two images were merged to count annexin V–positive cells (E and F). G: Summary bar graph for annexin V staining of cells transfected with control plasmid, wild-type CNGB3 plus CNGA3, or CNGB3-F525N plus CNGA3 plasmids. Fluorescent images were obtained using a Zeiss LSM 510 confocal system as described in the Methods. Scale bar in F (applies to A-F), 100 µm.|
|Figure 5. Effect of combined exposure to CPT-cAMP and CPT-cGMP on cytotoxicity of cells expressing channels with disease-associated mutations in CNGB3. A: Representative dose–response relationships for CPT-cAMP activation of CNG channels, after coexpression of CNGA3 with CNGB3-WT (circles), T383fsX (squares), or F525N (triangles) subunits (same representative patches as in Figure 1B). Currents were normalized to the maximum CPT-cGMP current. Continuous curves represent fits of the dose–response relationship with the Hill equation. Parameters for each channel type were as follows: for WT, K1/2,CPT-cAMP=28.3 μM and h=1.4; for T383fsX, K1/2, cAMP=27.9 μM and h=1.6; and for F525N, K1/2,CPT-cAMP=10.8 μM and h=1.0. B: Bar graph of the relative cytotoxicity for channel-expressing cells exposed to various concentrations of CPT-cAMP plus 0.1 μM CPT-cGMP (n=12). The dashed line represents the percentage of cell death in the vector-only control group without treatment; * indicates significant difference between groups designated by bracket (p<0.05); + represents significant difference between the F525N group treated with 10 μM CPT-cAMP together with 0.1 μM CPT-cGMP and the F525N group without treatment (p<0.01).|
|Figure 6. Block of CNG channels by L-cis-diltiazem increases viability of cells expressing CNGB3 with disease-associated mutations. A: Representative current traces elicited by 0.1 µM CPT-cGMP in the absence or presence (arrow) of 10 µM L-cis-diltiazem. Current traces were elicited by the voltage protocol described in the Methods section. B: Dose–response relationships for block by L-cis-diltiazem in the presence of 0.1 µM CPT-cGMP for heteromeric CNG channels containing CNGB3-WT (circles), T383fsX (squares), or F525N (triangles) subunits. Currents were normalized to the current elicited by 0.1 µM CPT-cGMP in the absence of L-cis-diltiazem. Continuous curves represent fits of the dose–response relation with the modified Hill equation described in the Methods section. Parameters for each channel type were as follows: WT, K1/2,L-cis-dilt.=4.1 µM and h=0.5; for T383fsX, K1/2,L-cis-dilt.=135 µM and h=0.3; and for F525N, K1/2,L-cis-dilt.=4.2 µM and h=0.7. C: Bar graph of the relative cytotoxicity for channel-expressing cells with or without 10 µM L-cis-diltiazem in the presence of 0.1 μM CPT-cGMP treatment (n=12). The dashed line indicates the extent of cell death in vector-only control cells without treatment. Significant differences were observed between groups indicated by brackets (*, p<0.05); + indicates significant difference between T383fsX groups treated with or without channel blocker (p<0.01).|
|Figure 7. Removal of extracellular Ca2+ from culture media prevented the increase in cytotoxicity for cells expressing CNGB3 F525N. Bar graph of the relative cytotoxicity for channel-expressing cells treated with 0.1 μM CPT-cGMP in normal or Ca2+-free Dulbecco’s modified Eagle’s medium media (n=12; *, p<0.05). The dashed line indicates the level of cytotoxicity in control cells expressing pOPRSVI plasmid alone cultured in normal media without CPT-cGMP.|
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