title: Inhibition of [3H]batrachotoxinin A-20alpha-benzoate binding to sodium channels and sodium channel function by endocannabinoids.
abstract: A number of putative endocannabinoids were found to modify the binding of [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]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 [(3)H]BTX-B binding with IC(50) values of 90.4, 51.2 and 20.7 microM, respectively. The CB1 receptor antagonist AM251 (2 microM) 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 [(3)H]BTX-B binding giving 14.8+/-2.2 and 23.9+/-4.8% inhibition at 100 microM. Phenylmethanesulphonylfluoride (PMSF) did not alter the inhibitory effects of 2-AG, AGE, NADA and A-Gly on binding, but the efficacy of 100 microM 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 [(3)H]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 [(3)H]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 occurred through a mechanism that was not influenced by 2 microM 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.
authors: Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada.
authors: Yin
authors: Y
authors: Duan
authors: Jian
authors: J
authors: Zheng
authors: Russell A
authors: RA
authors: Nicholson
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D001496:Batrachotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000493:pharmacokinetics; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects*; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D063385:Cannabinoid Receptor Modulators / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D005989:Glycerides / Q000494:pharmacology; D051379:Mice; D015394:Molecular Structure; D018377:Neurotransmitter Agents / Q000378:metabolism; D010599:Pharmacokinetics; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D011869:Radioligand Assay; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects*; D013574:Synaptosomes; D014316:Tritium
chemical_list: D001095:Arachidonic Acids; D001496:Batrachotoxins; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D005989:Glycerides; D018377:Neurotransmitter Agents; D043884:Receptor, Cannabinoid, CB1; D015222:Sodium Channels; D014316:Tritium; C031775:batrachotoxinin A 20-alpha-benzoate; C094503:glyceryl 2-arachidonate