The disease-causing mutations in the carboxyl terminus of the cone cyclic nucleotide-gated channel CNGA3 subunit alter the local secondary structure and interfere with the channel active conformational change

Abstract

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransducton. Mutations in the channel subunits are associated with achromatopsia and progressive cone dystrophy in humans. More than 50 mutations have been identified in the channel CNGA3 subunit, with 50% of them located in the carboxyl (C) terminus. This study investigates the defects of the two frequently occurring mutations, R377W and F488L, in the C-terminus of CNGA3. Ratiometric measurement of the intracellular Ca(2+) concentration and electrophysiological recordings showed the loss of functional activity of the mutant channels in an HEK293 heterologous expression system. Immunofluorescence labeling revealed an apparent cytosolic aggregation of the mutant channels compared to the wild type (WT). The R377W and F488L mutants, expressed and purified from Escherichia coli as glutathione S-transferase (GST) fused to the CNGA3 C-terminal domain, showed no negative effects on interactions with the channel subunits. Circular dichroism spectrum analyses were performed to examine the structural impact of the mutations. Although the R377W and F488L C-termini mutants retained stable, folded structures, the secondary structures of both mutants differed from the WT protein. Furthermore, the WT C-terminus exhibited a significant decrease in alpha-helical content in response to the channel ligands, while this allosteric transition was diminished in the two mutants. This is the first study showing the structural impact of the disease-causing mutations in the cone CNG channel subunit. The observed alterations in the local secondary structure and active conformational change may confer an adverse effect on the channel's activity and cellular processing.

Figure 1

Membrane topology of the mouse CNGA3 with locations of the R377W and F488L mutations indicated. CNBD is located between residues 429 and 545.

Figure 2

The R377W and F488L mutations cause loss of channel activity. (A). Intracellular calcium responses of HEK293 cells expressing the WT-CNGA3, R337W-CNGA3, and F488L-CNGA3 in response to 8-pCPT-cGMP (100 μM) stimulation. The left panel shows the representative response curves and the right panel is the bar graph showing the quantitative analysis of the calcium measurement (at 300 s after cGMP stimulation) from 4-5 independently performed experiments. (B). Electrophysiological recordings of HEK293 cells expressing the WT-CNGA3, R337W-CNGA3, and F488L-CNGA3 in response to 8-pCPT-cGMP (100 μM) stimulation. The recordings were performed using gap-free protocol with a holding potential at −50 mV. The left panel shows representative patch-clamp recording profiles and the right panel is the bar graph showing the quantitative analysis of peak amplitude in response to 100 μM 8-pCPT-cGMP (WT n = 18, R377W n = 13, and F488L n = 9). (C). Western blot detection of expression of the WT-CNGA3, R337W-CNGA3, and F488L-CNGA3 in HEK293 cells that had been transfected with the respective cDNAs. Cellular protein extractions were resolved on 10% SDS-PAGE, followed by Western blotting using the polyclonal anti-CNGA3 antibody.

Figure 3

The R377W and F488L mutations cause cytosolic aggregation of the channels. Immunofluorescence labeling was performed to determine the cellular localization of the WT-CNGA3, R337W-CNGA3, and F488L-CNGA3. (A). Representative confocal images showing cellular localization of the WT-CNGA3, R337W-CNGA3, and F488L-CNGA3 in HEK293 cells that had been transfected with the respective cDNAs. Scale bar: 10 μm. (B). The bar graph shows the quantitative results of the cellular fluorescence labeling intensities. Bars represent the means ± SEM of the number of cells (12 for the WT transfected, 18 for the R377W transfected, and 16 for the F488L transfected) from 3 independently performed experiments. Unpaired Student's t test was used for determination of the significance. *, p < 0.05.

Figure 4

The R377W and F488L mutations do not affect the channel subunit interactions. GST pull-down assays were performed to examine the subunit interactions and the effects of mutations. The GST fusion proteins bound to glutathione resin were incubated with Nrl^−/− retinal membrane extracts (∼100 μg protein) at 4°C for 2 h. The pull-down products were resolved on 10% SDS-PAGE, followed by Western blotting using antibodies as indicated. (A). Diagram of the GST-fusion proteins (left panel) and detection of their expression (right panel). (B). GST pull-down assays showing an involvement of the C-terminus of CNGA3 in the subunit interactions (left panels), and presence of a homo interaction of CNGA3 but not CNGB3 (right panels). (C). GST pull-down assays show that the R377W and F488L mutations do not affect the subunit interactions. The left panel shows expression of the GST fusion proteins of the WT, R377W, and F488L C-termini detected by Commassie blue staining, and the right panel is a representative pull-down result showing the WT and two mutant C-termini to interact with CNGA3 (upper panel) and CNGB3 (middle panel).

Figure 5

The R377W and F488L mutations result in decreased α-helicity of the C-terminus of CNGA3. CD spectra analyses were performed to examine the effects of the R377W and F488L mutation on the secondary structure of CNGA3. GST fusion proteins were expressed, purified and used in the CD analysis. (A). Representative CD spectra of the WT (black) and R377W (red) and F488L (green) mutants with subtraction of GST spectrum. The two mutants show CD spectra different from the WT. (B). Quantitative analyses of the amounts of α-helix and β-sheet were performed using three databases (Selcon, Contin, and CDSSTR). Shown are the summarized results of these analyses. The α-helical contents of the two mutants were significantly lower compared to the WT, where there were no differences in the amounts of β-sheet and random coil between WT and the two mutants. Unpaired Student's t test was used for determination of the significance (P < 0.05).

Figure 6

WT, but not the R377W and F488L mutants, shows a conformational change of the C-terminus in response to 8-pCPT-cGMP. (A). Representative CD profiles of the WT, R377W and F488L C-termini in response to varying concentrations (0, black; 3.0, blue; 10.0, green; 30.0, brown; and 100, red) (μM) of 8-pCPT-cGMP. A concentration-dependent reduction of negative ellipticity was seen in the WT but not the R377W and F488L mutants upon addition of 8-pCPT-cGMP. Representative CD spectral profiles of GST in response to varying concentrations of 8-pCPT-cGMP were included. (B). Quantitative analyses of α-helix and β-sheet amounts in the WT and mutant C-termini upon addition of 8-pCPT-cGMP (100 μM). Unpaired Student's t test was used for determination of the significance (P < 0.05).

Figure 7

WT, but not the R377W and F488L mutants, shows a conformational change of the C-terminus in response to cGMP and cAMP. Representative CD profiles of the WT, R377W and F488L C-termini in response to varying concentrations (0, black; 3.0, blue; 10.0, green; 30.0, brown; 100, red; and 300, pink) (μM) of cGMP (A) and cAMP (B). A concentration-dependent reduction of negative ellipticity was seen in the WT but not the R377W and F488L mutants upon addition of cGMP. cAMP at the highest concentration (300 μM) used induced a CD profile change in the WT but not the mutant C-termini. Representative CD spectral profiles of GST in response to varying concentrations of cGMP and cAMP were included.

Figure 8

The R377W and F488L mutations do not affect protein stability. (A). Thermal denaturation study of the WT and R377W and F488L mutant C-termini. Shown are thermal denaturation curves for the WT and mutant C-termini obtained at 220 nm from 20°C to 80°C (GST, black; WT, blue; R377W, green; and F488L, red). Denaturation curves are in units of molar ellipticity vs. temperature. The transition temperature was at ∼ 59°C for GST and was at ∼ 68°C for the C-terminal fusion proteins. The unfolding temperature at the R377W and F488L mutants was not different from that in the WT. (B). Limited tryptic digestion of the WT and R377W and F488L mutant CNGA3. The membranes prepared from cells that had been transfected with cDNAs encoding the WT and R377W and F488L mutants were incubated with trypsin-TPCK (30 μg/ml) at 30°C for 2, 5, and 10 min. The digested products were resolved on 10% SDS-PAGE, followed by Western blotting using the polyclonal anti-CNGA3 antibody. Shown are representative images showing limited proteolysis of the WT and mutant CNGA3 subunits. The cleavage rates of the mutants to trypsin were about the same as the WT.