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FIGURE 1. Voltage dependence of the potency of 2-AG. (A) and (B). Measurement of the relationship between 2-AG concentration and GIRK currents at -80 mV and at +40 mV, respectively. Basal GIRK current is evolved following replacement of the solution to a high K+ solution. Then, 4 different 2-AG concentrations (100, 1,000, 2000 and 10000 nM) were applied and the response for each concentration was measured. (C). Dose response curves for several 2-AG concentrations at −80 mV (black symbols and line; n = 19, 45, 51, 39, 9, 19 and 39 for 1, 10, 100, 1,000, 2000, 4,000 nM respectively) and at +40 mV (red symbols and line; n = 8, 26, 28, 15, 19, 12 and 39 for 1, 10, 100, 1,000, 2000, 4,000 nM respectively). The responses are normalized to the response evoked by 10000 nM 2-AG at each holding potential. (D) The dependence of the relative activation of the receptor by 100 nM 2-AG in voltage. Responses are normalized to the response evoked by 10000 nM 2-AG at each holding potential.
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FIGURE 2. Voltage dependence of the potency of AEA. (A) and (B). Measurement of the relationship between AEA concentration and GIRK currents at -80 mV and at +40 mV, respectively. Basal GIRK current is evolved following replacement of the solution to a high K+ solution. Then, 4 different AEA concentrations (1, 10, 400 and 10000 nM) were applied and the response for each concentration was measured. (C). Dose response curves for several AEA concentrations at −80 mV (black symbols and line; n = 31, 20, 20, 14, 25, 11, 8 and 41 for 1, 10, 100, 400, 1,000, 2000, 4,000, and 10000 nM, respectively) and at +40 mV (red symbols and line; n = 8, 10, 9, 8, 17, 11, 8 and 30 for 1, 10, 100, 400, 1,000, 2000, 4,000, and 10000 nM, respectively). The responses are normalized to the response evoked by 10000 nM AEA at each holding potential.
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FIGURE 3. Measurement of the dissociation of agonist from the CB1 receptor by measuring the deactivation of CB1 receptor-evoked GIRK current following agonist washout. (A) An example from one oocyte. Current evoked by 2-AG was deactivated by washout of the2-AG. The dashed red line represents an exponential fit to the decay of the current. (B) A comparison of the decay of GIRK currents evoked by AEA (black) or 2-AG (red) following washout of the agonist at time zero. The currents were normalized to enable comparison between the two recordings. (C) Results from 20 oocytes with each agonist. The time constant of the decay of AEA-evoked currents is significantly higher than that of 2-AG evoked currents.
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FIGURE 4. Voltage dependence of the dissociation of agonist from the CB1 receptor. (A) And (C)A comparison of the decay of GIRK currents at −80 mV (black) or at +40 mV (red). The currents were evoked following activation with 2-AG (A) or AEA (C). The washout of the agonists took place at time 0 and the currents were normalized to enable comparison between the two recordings. (B) and (D). Time constants of decay measured of −80 mV and +40 mV. The currents were evoked following activation with 2-AG (B) or AEA (D).Each two data points connected with a line represent the same oocyte at the two holding potentials.
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FIGURE 5. Voltage dependence of the potency of THC. (A) Dose response curves for several THC concentrations at −80 mV (black symbols and line; n = 29, 29, 29, 13, 12 and 30 for 1, 10, 100, 1,000, 5,000, 10000, and 50000 nM, respectively) and at +40 mV (red symbols and line; n = 18, 18, 18, 14, 8 and 21 for 1, 10, 100, 1,000, 5,000, 10000, and 50000 nM, respectively). The responses are normalized to the response evoked by 20 µM THC at each holding potential. (B) The dependence of the relative activation of the receptor by 100 nM THC in voltage. Responses are normalized to the response evoked by 50000 nM THC at each holding potential. (C) A comparison of the decay of GIRK currents at -80 mV (black) or at +40 mV (red). The washout of the agonists took place at time 0 and the currents were normalized to enable comparison between the two recordings. (D) Time constants of decay measured of −80 mV and +40 mV. Each two data points connected with a line represent the same oocyte at the two holding potentials.
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Figure S1. Effect of 2-AG on GIRK channels. (A) A representative recording from an oocyte expressing the GIRK channel. Application of 10 µM 2-AG did not evoke GIRK currents or inhibited basal GIRK currents. (B) Current -Voltage (I-V) curve of IK before (black) and after (red) the application of 2-AG. The oocytes were voltage clamped to -80 mV and the currents after depolarizing pulses to various holding potentials from –80 mV to +40 mV at 10 mV increments were measured. Data is mean ± SEM from 7 oocytes. The difference between the two conditions is not significant (paired t-test, p>0.11 for all voltages).
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Figure S2. Effect of AEA on GIRK channels. (A) A representative recording from an oocyte expressing the GIRK channel. Application of 10 µM AEA did not evoke GIRK currents or inhibited basal GIRK currents. (B) Current-voltage I-V curve of IK before (black) and after (red) the application of AEA. The oocytes were voltage clamped to -80 mV and the currents after depolarizing pulses to various holding potentials from –80 mV to +40 mV at 10 mV increments were measured. Data is mean ± SEM from 7 oocytes. The difference between the two conditions is not significant (paired t-test, p>0.13 for all voltages).
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Figure S3. Effect of THC on GIRK channels. (A) A representative recording from an oocyte expressing the GIRK channel. Application of 10 µM THC did not evoke GIRK currents or inhibited basal GIRK currents. (B) I-V curve of IK before (black) and after (red) the application of THC. The oocytes were voltage clamped to -80 mV and the currents after depolarizing pulses to various holding potentials from –80 mV to +40 mV at 10 mV increments were measured. Data is mean ± SEM from 5 oocytes. The difference between the two conditions is not significant (paired t-test, p>0.23 for all voltages).
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Figure S4. I-V curve of IK before (empty circles) and after the application of 100 and 10000 nM 2-AG (black and red symbols, respectively), measured from one oocyte. The oocyte was voltage clamped to -80 mV and the currents after depolarizing pulses to various holding potentials from –80 mV to +40 mV at 10 mV increments were measured.
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