Inhibition of [3H]batrachotoxinin A-20α-benzoate binding to sodium channels and sodium channel function by endocannabinoids

A number of putative endocannabinoids were found to modify the binding of [3H]batrachotoxinin A-20α-benzoate ([3H]BTX-B) to site 2 on voltage-gated sodium channels of mouse brain and achieve functional inhibition of sodium channels in vitro. 2-Arachidonoyl-glycerol (2-AG), arachidonoyl glycerol ether (AGE), N-arachidonoyl-dopamine (NADA) gave almost complete inhibition of [3H]BTX-B binding with IC50 values of 90.4, 51.2 and 20.7 μM, respectively. The CB1 receptor antagonist AM251 (2 μM) had no effect on the displacement of radioligand by these endocanabinoids. Arachidonoyl-glycine (A-Gly) and arachidonoyl-GABA (A-GABA) were apparently less effective inhibitors of [3H]BTX-B binding giving 14.8 ± 2.2 and 23.9 ± 4.8% inhibition at 100 μM. Phenylmethanesulphonylfluoride (PMSF) did not alter the inhibitory effects of 2-AG, AGE, NADA and A-Gly on binding, but the efficacy of 100 μM A-GABA was increased by 60.3 ± 6.3% (P < 0.05). Scatchard analyses showed that 2-AG, AGE and NADA reduce the binding of [3H]BTX-B by lowering Bmax although increases in KD were also evident for AGE and NADA. Our kinetic experiments found that 2-AG, AGE and NADA increase the dissociation velocity of radioligand from site 2 on sodium channels demonstrating that these endocannabinoids operate as allosteric inhibitors of [3H]BTX-B binding. 2-AG, AGE and NADA inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes at low micromolar concentrations and again the capacities of A-Gly and A-GABA to inhibit this response were less pronounced. The three most effective endocannabinoids (2-AG, AGE and NADA) were then examined in a synaptosomal transmitter release assay where they were observed to inhibit sodium channel- (veratridine-dependent) release of l-glutamate and GABA in the low micromolar range. These effects also occured through a mechanism that was not influenced by 2 μM AM251. It is concluded that direct inhibition of sodium channel function leading to reduced neuronal excitation and depression of presynaptic release of amino acid transmitters is a property shared by several endocannabinoids.