Figure 1. Effect of CBD on activation of holo-HCN4 channels. (A) Representative voltage protocol and activation current traces for mHCN4 construct before (black) and after (green) treatment with 5 µM CBD. The −100 mV trace is highlighted. (B) Average tail current values for treatment for mHCN4 before (black) and after application of 5 µM CBD (green). Values fit to a Boltzmann curve. Average V_1/2 for mHCN4 was −102.1 ± 1.1 (n = 9); average V_1/2 for 5 µM CBD was −87.5 ± 3.0 mV (n = 9). (C) Scatterplot of a shift in average ΔV_1/2 ± SEM for the vehicle (black) and increasing concentrations of CBD (green). One-way ANOVA and post-hoc Tukey’s test found a significant shift for 5, 10, and 20 µM CBD (***) versus the vehicle (P < 0.0001) and 1 µM CBD (P < 0.03). (D) ΔV_1/2 of activation after treatment with various concentrations of CBD. Data were fit with the Hill equation. EC₅₀ was 1.59 ± 0.21 µM with a slope of 2.50 ± 0.63. (E) Relative peak current from the end of activation epoch (I_{post-treatment}/I_{pre-treatment}) for the vehicle (black) and increasing concentrations of CBD (green). One-way ANOVA and post-hoc Tukey’s test found no significant differences in relative current levels after treatments (P > 0.2). More about this image found in Effect of CBD on activation of holo-HCN4 channels. (A) Representative volt...

Figure 2. Effect of CBD on activation kinetics of holo-HCN4 channels. (A) Superimposition of activation epoch (−120 mV) pre- (black) and post-treatment with 5 µM CBD (green), dotted line shows 0 current. Single exponential fit of activation epochs shown in dashed lines. (B) Average activation τ ± SEM between −140 and −100 mV. One-way ANOVA and post-hoc Tukey’s test found all concentrations of CBD significantly sped activation compared with vehicle (*P < 0.05; **P < 0.01; ***P < 0.001). Additionally at −120 mV, treatment with 5–20 µM CBD significantly sped activation compared to treatment with 1 µM CBD (P < 0.02). (C) Use-dependence 1/3 Hz protocol at −120 mV pre (black) and posttreatment with 5 µM CBD (green), sweep 1, 5, and 10 shown. The effect of CBD does not change over time. Dotted line shows 0 current. (D) Average τ_act ± SEM at −120 mV during 1/3 Hz protocol (n = 7), total of 20 sweeps. Unpaired Student’s t test found CBD significantly sped activation (P < 0.03), but that after treatment with 5 µM CBD sweep 1 τ_act is not significantly different than sweep 20 τ_act (P > 0.2). Thus, the effect of CBD does not change over the course of 60 s. More about this image found in Effect of CBD on activation kinetics of holo-HCN4 channels. (A) Superimpos...

Figure 3. CBD potentiation is distinct from cAMP and PIP ₂ . (A) Representative voltage protocol and traces of mHCN4 in the absence of cAMP before (black) and after (purple) treatment with 5 µM CBD. The −100 mV trace is highlighted. (B) Average tail current values for pre- (black square) and post-treatment with 5 µM CBD (purple square) in the absence of cAMP (apo-HCN4 channels), and treatment with 5 µM CBD in the presence of cAMP (green circle). Values were fit to the Boltzmann function. Average V_1/2 for pre-treatment (black square) was −113.2 ± 2.2 mV (n = 7); average V_1/2 for post-treatment with 5 µM CBD (purple square) was −97.6 ± 2.5 mV (n = 7); average V_1/2 for post-treatment with 5 µM CBD in presence of cAMP (green circle) was −87.5 ± 3.0 mV (n = 9). One-way ANOVA and post-hoc Tukey’s test found that the application of CBD significantly shifted V_1/2 (P < 0.003) by ∼15 mV, and the presence of cAMP and CBD significantly shifted V_1/2 (P < 0.04) a further 10 mV. (C) Representative traces of mHCN4 (black) and mHCN4 after application of diC8-PI(4,5)P₂ (orange). The −100 mV trace is highlighted. (D) Average tail current values for treatment with vehicle (black circle), 5 µM CBD (green circle), and 5 µM CBD with 10 µM diC8-PI(4,5)P₂ (orange diamond). Values fit to the Boltzmann function. More about this image found in CBD potentiation is distinct from cAMP and PIP ₂ . (A) Repres...

Figure 4. Computed highest binding affinity binding site of CBD with the cAMP bound HCN4 structure. (A) Space-fill model of HCN4, CBD (green) highlighted by green circle. (B) Zoomed in view of lipid binding pocket bound by CBD. (C) CBD binding location in the VSD of one subunit. (D) CBD is... More about this image found in Computed highest binding affinity binding site of CBD with the cAMP bound H...

Figure 5. Schematic for the mechanism of action of CBD (green) on HCN4 channels. Top: Under normal physiological conditions, HCN4 channels would not open at weak driving forces (i.e., −80 mV). Bottom: When CBD is added, it stabilizes the S4 in the activated state, allowing HCN4 channels to open ... More about this image found in Schematic for the mechanism of action of CBD (green) on HCN4 channels. Top...

Figure 1. Substitution of the critical amino acid S42 at the C-terminal end of the pore-helix by threonine has dramatic impacts on gating and K ⁺ conductance of Kcv _NTS . (A) Selectivity filter and pore helix of Kcv_NTS (grey, homology model on KirBac1.1 [PDB ID 1P7B ] calculated with Swiss model), KcsA (blue, PDB ID 1K4C ), and MthK (pink, PDB ID 3LDC ). Some of the hydrogen bonds are drawn in red for KcsA and MthK (as identified by UCSF chimera) with the red spheres being buried water molecules. (B) Representative single-channel current traces recorded in DPhPC bilayers at constant voltages between −160 and +160 mV from Kcv_NTS and Kcv_NTS S42T. The closed and open levels are marked by C and O, respectively. (C and D) Current–voltage relationships and open probabilities (P_O) from time series like those in B. Symbols as in B. Data points show arithmetic mean ± SD (often hidden under the data points) of at least three independent measurements. More about this image found in Substitution of the critical amino acid S42 at the C-terminal end of the po...

Scheme 1 More about this image found in Untitled image

Figure 2. Characteristics of voltage-dependent closure of Kcv _NTS S42T show large variability between different sets of experiments. (A) Representative Kcv_NTS S42T single-channel current traces recorded in DPhPC bilayers from two independent sets of experiments performed over several months. Applied voltages are depicted on the left. Closed and open levels are marked by C and O, respectively. (B) Open probabilities from time series like those in A. Data points correspond to symbols in A and show arithmetic mean ± SD of at least three independent measurements. P_O/V relationships are well described by a single Boltzmann equation (solid lines, Eq. 1 ) with V_1/2 of 119 ± 7 and 155 ± 3 mV for experiments 1 and 2, respectively; the corresponding gating valences (δz) are −0.97 ± 0.04 e₀ and −0.73 ± 0.16 e₀ for experiments 1 and 2, respectively. More about this image found in Characteristics of voltage-dependent closure of Kcv _NTS S42T s...

Figure 3. Inactivation of Kcv _NTS S42T at positive potentials is caused by a voltage-dependent increase in the frequency of closings and the appearance of two voltage-dependent closed-time populations. (A) Representative open (top) and closed (bottom) dwell-time histograms from single-channel recordings of Kcv_NTS S42T. Test voltages as well as a short current trace are reported above corresponding dwell-time histograms. Open dwell-time histograms show only one open dwell-time population (O) and were therefore fitted with a single exponential function (solid red line). Closed dwell-time histograms were fitted with three or two exponential functions. Closed dwell-time populations are indicated with C1 (solid green line), C2 (solid blue line) and C3 (solid yellow line). The sums of these exponentials are shown as solid black lines. The number of detected events used to construct the closed and open dwell-time histograms are indicated in the open time histograms. (B) Voltage-dependence of mean open (red) and mean closed (black) lifetimes calculated from dwell-time histograms as in A. (C) Mean lifetimes of closed dwell-time populations C2 (blue) and C3 (yellow) calculated from dwell-time histograms as in A. (D) Voltage dependence of occupation probabilities of open state O (red) and closed-time populations C1 (green), C2 (blue), and C3 (yellow). Data points in B–D show geometric mean ± geometric SD of three independent measurements. (E) Representative single-channel current traces from Kcv_NTS and Kcv_NTS S42T at high voltages of +220 and +300 mV. Closed and open levels are marked by C and O, respectively. (F) Open probabilities from time series like in E. Data points correspond to symbols in E and show arithmetic mean ± SD of at least three independent measurements. The solid red line shows the P_O/V relationship expected for voltage-dependent gating based on the Boltzmann equation ( Eq. 1 ). More about this image found in Inactivation of Kcv _NTS S42T at positive potentials is caused ...

Figure 4. Screening of divalent cations reveals that mutation S42T increases sensitivity to the blocking ions Sr ²⁺ and Ba ²⁺ . (A) Representative single-channel current traces recorded in DPhPC bilayers at +120 mV from Kcv_NTS and Kcv_NTS S42T in the absence (top row) or presence of 100 µM of the divalent cations Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺ in the cytosolic solution. The closed and open levels are marked by C and O, respectively. (B) Inhibitory effect of 100 µM cytosolic Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺ on the open probability of Kcv_NTS and Kcv_NTS S42T calculated from single channel recordings as in A. (C and D) Voltage-dependence of the inhibitory effect of 10 μM Sr²⁺_cyt (C) or 1 μM Ba²⁺_cyt (D) on the open probability of the Kcv_NTS (closed squares) and the S42T mutant (open squares). Data points in B–D show arithmetic mean ± SD of three independent measurements. More about this image found in Screening of divalent cations reveals that mutation S42T increases sensitiv...