Journal: bioRxiv

Article Title: HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

doi: 10.1101/2020.03.02.973560

Figure Lengend Snippet: HCND is essential for the surface expression of HCN channels. a , Representative Immunoblot of the biotinylated protein fraction (upper half) for untransfected (control), WT and ΔHCND mHCN2 transfected HEK 293 cells probed with HCN2 specific antibodies. Representative immunoblot of the whole-cell lysates (lower half) from untransfected (control), WT and ΔHCND mHCN2 transfected HEK 293 cells probed with α-tubulin antibodies. b , Quantification of unglycosylated channel expression in untransfected (control), WT and ΔHCND mHCN2 transfected HEK 293 cells. n = 3.

Article Snippet: The HCN channels were detected using a rabbit anti-HCN2 primary antibody (Boster, A02804) that recognizes the C-terminal sequence of HCN2 channels and a goat anti-rabbit secondary antibody (Abcam, ab6721).

Techniques: Expressing, Western Blot, Control, Transfection

Journal: bioRxiv

Article Title: HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

doi: 10.1101/2020.03.02.973560

Figure Lengend Snippet: Interactions between the HCND and C-linker/CNBD affect voltage-dependent gating of HCN2 channels. a , Enlarged view of the interaction interface between the HCND and the C-linker/CNBD in HCN1 channels. The interacting residues E436 and Q440 on the C-linker, H517 on the CNBD and R112 on the HCND are shown as sticks. b , Representative currents from E478A_Q482A_H559A (3M) mutant mHCN2 channels. E478, Q482 and H559 correspond to E436, Q440 and H517 in HCN1 channels. c , Averaged conductance-voltage relationship for the currents from WT (black circles, n of 9) and 3M mutant (red circles, n of 10) mHCN2 channels. Lines correspond to the fits with the Boltzmann function with the V 1/2 of −91.3 ± 0.3 mV for WT and −97.1 ± 0.3 mV for the 3M mutant, and s of 13.8 ± 0.2 and 11.9 ± 0.3 for 3M mutant mHCN2 channels.

Article Snippet: The HCN channels were detected using a rabbit anti-HCN2 primary antibody (Boster, A02804) that recognizes the C-terminal sequence of HCN2 channels and a goat anti-rabbit secondary antibody (Abcam, ab6721).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

doi: 10.1101/2020.03.02.973560

Figure Lengend Snippet: Interactions between the HCND and C-linker/CNBD affect cAMP-dependent gating of HCN2 channels. Representative currents from WT ( a ) and 3M mutant ( b ) mHCN2 channels recorded at −90 mV in the absence (black) and presence (red) of 10 μM cAMP. c , Plots of the percent increase in tail currents versus the cAMP concentration for WT (black circles) and 3M (red circles) mutant mHCN2 channels. The tail currents were recorded at −40 mV after the test pulse to −90 mV. Lines correspond to the fits with the Hill equation with the Kd of 0.1 ± 0.04 μM for WT and 0.06 ± 0.2 μM for 3M mutant mHCN2 channels. n ≥ 5 for each condition. d , Plots of deactivation time constants for tail currents recorded at −40 mV after the test pulse to −90 mV for WT and 3M mutant channels in the presence and absence of 10 μM cAMP, as indicated. n = 6 for WT and n = 5 for 3M mutant channels. The data with and without cAMP for WT and 3M channels were compared using paired Student t-test. The data between WT and 3M mutant channels were compared using unpaired Student t-test. * P < 0.02.

Article Snippet: The HCN channels were detected using a rabbit anti-HCN2 primary antibody (Boster, A02804) that recognizes the C-terminal sequence of HCN2 channels and a goat anti-rabbit secondary antibody (Abcam, ab6721).

Techniques: Mutagenesis, Concentration Assay