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

Abstract

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 IC 50 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 B max although increases in K D 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.