Mayar S , Memarpoor-Yazdi M , Makky A , Eslami Sarokhalil R , D'Avanzo N .

             cannabinoid signaling pathway
             HCN channel complex

	FIGURE 1. Cannabinoid regulation of Ih in neurons. The synthetic cannabinoid WIN55,212-2 affects memory via increasing Ih by activating CB1 receptors, leading to JNK-mediated increases in activated nitric oxide synthase (NOS), resulting in increased guanylyl cyclase activity and, in turn, cGMP (Hill et al., 2012). However, cannabinoids have also been shown to modify the function of several ion channels independently of cannabinoid receptors (CBRs) activation. Here, we ask if hyperpolarization-activated cyclic-nucleotide-gated (HCN1) channels can be directly modulated by cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) (Maroso et al., 2016).
	FIGURE 2. Concentration-dependent regulation of HCN1 current by THC and CBD. (A) Steady-state current values from a representative cell following repetitive pulses to −130 mV from VH = 0 mV with the addition of 0, 10, 20, 30, and 50 μM CBD. (B) Concentration dependence of HCN1 activation by CBD (■) at −130 mV (n = 13). This activation of HCN1 persists in the presence of the CB1R antagonist 10 μM AM-251 (∘) (n = 5). A total of 50% Max response (EC50) for CBD is elicited at 28.5 μM, with a 91% maximal increase in current. (C) CBD has no effects on uninjected oocytes, indicating that we are not observing effects on background currents. (D) CBD activated currents can be fully inhibited by 500 μM ZD7288. This indicates that CBD is only activating HCN1 currents, and not activating a background current in oocytes. (E) Steady-state currents from a representative cell following repetitive pulses to −130 mV in 0, 10, 25, and 50 μM THC. (F) Steady-state currents from a representative cell following repetitive pulses to −130 mV in the presence of the quantity of methanol used as a vehicle for 0, 10, 25, and 50 μM CBD/THC. (G) Concentration dependence of HCN1 inhibition by THC (■) or methanol (□) at −130 mV (n = 5). THC induces a 63% maximal block of HCN1 currents, with a half-maximal response (Relative IC50) of 21.8 μM. 50% block of total current (Absolute IC50) occurs at 28.9 μM. Methanol induces <a 5% decrease in current at concentrations above 20 μM (n = 4).
	FIGURE 3. Regulation of HCN1 by cannabidiol (CBD) and Δ9-tetrahydrocannabidiol (THC). (A) Representative traces from a paired experiment following the addition of increasing concentrations of CBD to oocytes expressing full-length HCN1. (B) Current–voltage (I/V) relationship in presence of CBD normalized to maximal current (IWT(−160mV)) (5 < n < 18 per condition; P < 0.05 for 30–50 μM). (C) Steady state activation in presence of CBD (P = 0.81 for V1/2). (D) Activation time constant (τ) kinetics in presence of CBD (0.21 < P < 0.71). (E) Deactivation time constant (τ) kinetics in presence of CBD (3 < n <7 per condition; 0.09 < P <0.65). (F) Representative traces from a paired experiment following the addition of increasing concentrations of THC to oocytes expressing full-length HCN1. (G) (I/V) relationship in presence of THC normalized to maximal current (IWT(−160mV)) (4 < n <8 per condition; P <0.05 for Gmax (slope between −120 and −160 mV) of 20–50 μM). (H) Steady-state activation in presence of THC (P = 0.49 for V1/2). (I) Activation time constant (τ) kinetics in presence of THC (0.21 < P < 0.90). (J) Deactivation time constant (τ) kinetics in presence of THC (3 < n < 8 per condition; 0.11 < P < 0.45).
	FIGURE 4. Effects of CBD and THC on HCN1 hysteresis. (A) Ramps from 0 to −150 mV and back to 0 mV at speeds of 600, 300, 150, 75, and 37.5 mV/s. The degree of hysteresis was quantified by the area between the forward and backward current traces when plotted vs. the membrane voltage. (B) Histogram of HCN1 hysteresis for HCN1 treated with different concentrations of methanol used as vehicles for the cannabinoids at concentrations listed in parentheses. Hysteresis is unaffected by increasing concentrations of methanol. (C–E) THC reduces the degree of hysteresis at fast ramp speeds, with a greater range of ramp speeds affected as THC concentration increases. CBD did not affect HCN1 hysteresis (3 < n < 10 for each condition; *P < 0.05).
	FIGURE 5. Effects of CBD and THC on HCN1 do not require the CNBD. (A) Current-voltage (I/V) relationship of HCN1ΔCNBD in the presence of increasing concentrations of CBD normalized to maximal current (IWT(−160mV)) (4 < n < 13 per condition; P < 0.05 for 20–50 μM). (B) Deactivation time constant (τ) kinetics of HCN1ΔCNBD following treatment with CBD (4 < n < 10 per condition; P < 0.05 for 20–50 μM). (C) Current-voltage (I/V) relationship of HCN1ΔCNBD in the presence of increasing concentrations of THC normalized to maximal current (IWT(−160mV)) (4 < n <12 per condition; P < 0.05 for 10–50 μM). (D,E) THC reduces the degree of HCN1ΔCNBD hysteresis at fast ramp speeds, with a greater range of ramp speeds affected as THC concentration increases. CBD did not affect HCN1ΔCNBD hysteresis (3 < n <10 for each condition; *P < 0.05).
	FIGURE 6. Changes in membrane fluidity do not account for cannabinoid regulation of HCN1. Membrane fluidity was altered by the addition of Triton X-100 (TX-100) to the bath solution. (A) Representative time course of the steady-state current following the application of TX-100 at different concentrations, indicating that increasing membrane fluidity decreases HCN1 channel activity. However, this decrease in HCN1 current occurs more rapidly (within 1–2 min) than the decline observed with THC (which occurs over the course of 10–30 min). (B) Current-voltage relationship of HCN1 upon the addition of 0, 15, 25, 30, and 45 μM TX-100 normalized to maximal current (IWT(−160mV)). (C) TX-100 induces a −10 to −15 mV hyperpolarizing shift in the steady-state voltage-dependence of HCN1, which does not occur when THC or CBD is applied. (D) HCN1 activation kinetics are uniquely affected by TX-100, with membrane fluidity having a greater impact on slowing channel activation with more hyperpolarization of the membrane potential (3 < n < 9 per condition; P < 0.05).
	Supplementary Figure 1. Regulation of full-length HCN1 and HCN1-CNBD by methanol (MeOH). (A) Current-voltage (I/V) relationship of full-length HCN1 in presence of MeOH normalized to maximal current (IWT (-160 mV)). MeOH quantities used were equivalent to the amounts used as a vehicle during the addition of the concentration of cannabinoids shown in brackets (4 < n < 10 per condition; 0.45 < P < 0.87 Gmax (slope between -120mV and -160mV)). (B) Steady-state activation of full-length HCN1 in presence of MeOH. (P = 0.14 for V1/2). (C) Activation time constant () kinetics of full-length HCN1 in presence of MeOH. (0.12 < P < 0.87). (D) Deactivation time constant () kinetics of full-length HCN1 in presence of MeOH. (n = 3-4 per condition; 0.23 < P < 0.83). (E) Current-voltage (I/V) relationship of HCN1-CNBD in presence of MeOH normalized to maximal current (IWT (-160 mV)). (4 < n < 11 per condition; 0.16 < P < 0.93) (F) Steady-state activation of HCN1-CNBD in presence of MeOH. (P = 0.89 for V1/2) (G) Activation time constant () kinetics for of HCN1-CNBD in presence of MeOH. (H) Deactivation time constant () kinetics of HCN1-CNBD in presence of MeOH. (4 < n < 6 per condition; 0.23 < P < 0.63)
	Supplementary Figure 2. Regulation of HCN1-CNBD by cannabidiol (CBD) and 9-tetrahydrocannabidiol (THC). (A) Current-voltage (I/V) relationship in presence of CBD normalized to maximal current (IWT (-160 mV)) (4 < n < 13 per condition; P < 0.05 for 20 - 50 M). (B) Steady state activation in presence of CBD (P = 0.63 for V1/2). (C) Activation time constant () kinetics in presence of CBD (0.11 < P < 0.46) (D) Deactivation time constant () kinetics in presence of CBD (4 < n < 10 per condition; P < 0.05 for 20-50 M). (E) Current-voltage (I/V) relationship in presence of THC normalized to maximal current (IWT (-160 mV)) (4 < n < 12 per condition; P < 0.05 for 10 - 50 M). (F) Steady state activation in presence of THC. (P = 0.34 for V1/2). (G) Activation time constant () kinetics in presence of THC (0.12 < P < 0.46). (H) Deactivation time constant () kinetics in presence of THC (4 < n < 11 per condition; 0.23 < P < 0.84).
	Supplementary Figure 3. Activation of TRPV1 by CBD. (A) Representative traces from a paired experiment following the addition of increasing concentrations of CBD to oocytes expressing full-length rTRPV1. Membrane voltage was stepped from a holding potential of 0 mV to –80 to 170 mV. 10 M Capsaicin was added at the end of each experiment. (B) Normalized current voltage relationship (I/I(Control +170 mV)) after addition of increasing concentrations of CBD shown (n=8; P < 0.05 for Gmax (slope between +120 and +170 mV) of 20 CBD, 30 M CBD and Cap vs. Control).

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