Supplementary Figure 6: Impact of de novo HCN1 mutations on protein expression and localization at the plasma membrane.

(a) Semiquantitative analysis of WT and mutant (S100F, S272P, R297T, H279Y or D401H) HCN1 protein expression in whole-cell lysates and at the plasma membrane by protein blot. CHO-K1 cells were transiently cotransfected with 12 μg of WT or mutant HCN1 expression plasmid using the Neon electroporation system (Invitrogen); 24 h after transfection, proteins present at the plasma membrane were isolated using the Cell Surface Protein Isolation kit (Pierce), following the manufacturer's recommendations. Whole-cell lysates were kept before isolation of membrane protein from the same experiments. Proteins present in both fractions were resolved by SDS-PAGE on 4–12% gradient gels (Invitrogen) and electrotransferred onto nitrocellulose membranes. HCN1 was probed using a monoclonal mouse anti-HCN1 antibody (ab84816, Abcam; 1:10,000 dilution), and the signal was visualized with enhanced chemiluminescence (Pierce). Membranes were probed with anti–Flotillin-1 antibody (610820, BD Biosciences; 1:1,000 dilution) for normalization. The image shows the result of a representative experiment. (b) Semiquantitative measurement of WT and mutant HCN1 proteins present in whole-cell lysates (blue) and at the plasma membrane (red) from three independent experiments using the ImageJ program (http://rsb.info.nih.gov/ij/). The values obtained for WT and mutant HCN1 proteins were corrected for the intensity of the corresponding flotillin bands. Data are presented as means ± s.e.m. Note that, although the expression of S100F, S272P and R297T HCN1 channels was similarly decreased, an I_h current was recorded for S100F but nor for S272P and R297T. In addition, the S272P and R297T mutants showed a dominant-negative effect on the WT-mutant heteromeric channels, suggesting that mutant homomeric channels are less stable than WT-mutant heteromeric channels. (c) Effect of proteasome inhibition on mutant (S100F, S272P, R297T, H279Y or D401H) HCN1 protein expression in whole-cell lysates. CHO-K1 cells were transiently cotransfected with WT or mutant HCN1 expression plasmids; 24 h after transfection, half of the cells was treated with 1 μM epoxomicin (E3652, Sigma) for 16 h. Proteins were then resolved by SDS-PAGE on 4–12% gradient gels (Invitrogen) and electrotransferred onto nitrocellulose membranes. HCN1 was probed using the monoclonal mouse anti-HCN1 antibody, and the signal was detected by enhanced direct near-infrared fluorescence detection system (LI-COR Biosciences). Membranes were probed with anti-actin antibody (ab3280, Abcam; 1:4,000 dilution) for normalization. The image shows the result of a representative experiment.