|
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, â80 mV, and then back to 0 mV. B: Representative doseâresponse 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 doseâresponse 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 Vâpositive 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 Vâpositive 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 doseâresponse 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 doseâresponse 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: Doseâresponse 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 doseâresponse 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 Dulbeccoâs modified Eagleâs 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.
|