title: Complex association and dissociation kinetics of brevetoxin binding to voltage-sensitive rat brain sodium channels.
abstract: Brevetoxins bind with high affinity to the voltage-sensitive sodium channel and cause nerve cell depolarization and increased sodium ion conductance. Using 0.6 nM tritium-labeled brevetoxin-3 and freshly prepared synaptosomes from fresh or frozen rat brain, binding results at 6 degrees C fit well to a curve for 2-phase association with 65% of the binding in the rapid phase and t1/2 values of 11 and 74 min for the rapid and slow phases, respectively. Both phases were accelerated at higher toxin concentrations, binding of 9 nM brevetoxin-3 (PbTx-3) was close to equilibrium within 1 hr. The slow phase was not apparent when binding was done at 20 degrees C or when binding was done at 6 degrees C after the membrane sample had been preincubated at 4 degrees C for 1 day or at 22 degrees C for 1 hr. The 2-phase nature of association was not affected by substitution of KCl for choline chloride in the assay medium to produce sodium channel in the inactive state. Dissociation kinetics at 6 degrees C were also complex; the results fit well to a 2-phase curve with 55% of the dissociation in the rapid phase and t1/2 values of 13 and 64 min for the rapid and slow phases, respectively. The 2-phase nature did not change significantly after preincubation at 4 degrees C for 1 day. However, dissociation at 20 degrees C was rapid and fit a curve for 1-phase dissociation with a t1/2 of 2-6 min. At higher concentrations of PbTx-3, the binding is further complicated by the presence of 2-4 low-affinity binding sites with Kd values near 700 nM. In conclusion the association and dissociation of PbTx-3 with sodium channel from rat brain are complex processes that may involve changes in sodium channel conformation or interactions with other membrane (or membrane-associated) components.
authors.affiliation: NIEHS Marine and Freshwater Biomedical Sciences Center, University of Miami RSMAS, Florida 33149, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Whitney
authors.forename: D G
authors.initials: DG
authors.lastname: Baden
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D007700:Kinetics; D008387:Marine Toxins / Q000378:metabolism*; D035964:Oxocins; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000378:metabolism*; D000095702:Polyether Toxins
chemical_list: D008387:Marine Toxins; D035964:Oxocins; D015222:Sodium Channels; C053342:brevetoxin; D000095702:Polyether Toxins