title: pH-dependent binding of local anesthetics in single batrachotoxin-activated Na+ channels. Cocaine vs. quaternary compounds.
abstract: The effects of internal and external pH on the binding kinetics of local anesthetics (LAs) were studied in single batrachotoxin-activated Na+ channels incorporated into planar bilayers. With internal quaternary QX-314 and RAC421-II drugs, the binding interactions were little affected by either external or internal pH. With tertiary cocaine, the binding kinetics were drastically altered by pH. A decrease in the internal pH from 9.3 to 6.2 decreased the apparent equilibrium dissociation constant (Kd) of internal cocaine by more than 100-fold. This increase in the binding affinity was mostly accounted for by an increase in the apparent cocaine on-rate constant (kon) of approximately 80-fold. The cocaine off-rate constant (koff) was little changed (between 3-4 s-1). These results demonstrate quantitatively that the charged form of cocaine is the active form for BTX-activated Na+ channels. Surprisingly, the apparent pKa of cocaine near its binding site was estimated to be 1.4 units lower than that in bulk solution (7.1 vs. 8.5), indicating that the LA drug encounters a relatively hydrophobic environment. Opposite to the internal pH effect, a decrease of external pH from 8.4 to 6.2 increased the Kd value of internally and externally applied cocaine by approximately 8- and approximately 25-fold, respectively. External pH effect was primarily mediated by modulation of kon; koff was again relatively unaffected. Our findings support a model in which neutral cocaine can readily cross the membrane barrier, but needs to be protonated internally to bind to its binding site.
authors.affiliation: Department of Anesthesia Research Laboratories, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: J
authors.initials: J
authors.lastname: Nettleton
authors.forename: G K
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000779:Anesthetics, Local / Q000378:metabolism*; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D001665:Binding Sites; D003042:Cocaine / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008012:Lidocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D008051:Lipid Bilayers; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011485:Protein Binding; D011760:Pyrrolidinones / Q000378:metabolism*; D011817:Rabbits; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
chemical_list: D000779:Anesthetics, Local; D001496:Batrachotoxins; D008051:Lipid Bilayers; D009281:Naphthalenes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011760:Pyrrolidinones; D015222:Sodium Channels; C012647:QX-314; C065386:RAC 421-II; D008012:Lidocaine; D003042:Cocaine