title: Effect of depolarization on binding kinetics of scorpion alpha-toxin highlights conformational changes of rat brain sodium channels.
abstract: Binding of scorpion alpha-toxins to receptor site 3 on voltage-gated sodium channels inhibits sodium current inactivation and is voltage-dependent. To reveal the direct effect of depolarization, we analyzed binding kinetics of the alpha-toxin Lqh-II (from Leiurus quinquestriatus hebraeus) to rat brain synaptosomes and effects on rat brain II (rBII) channels expressed in mammalian cells. Our results indicated that the 33-fold decrease in toxin affinity for depolarized (0 mV, 90 mM [K(+)](out), K(d) = 5.85 +/- 0.5 nM) versus polarized (-55 mV, 5 mM [K(+)](out), K(d) = 0.18 +/- 0.04 nM) synaptosomes at steady state results from a 48-fold reduction in the association rate (k(on) at 5 mM [K(+)] = (12.0 +/- 4) x 10(6) M(-1) s(-1) and (0.25 +/- 0.03) x 10(6) M(-1) s(-1) at 90 mM [K(+)](out)) with nearly no change in the dissociation rate. Electrophysiological analyses of rBII channels expressed in mammalian cells revealed that approximately 75% and 40% of rBII occupied fast- and slow-inactivated states, respectively, at resting membrane potential of synaptosomes (-55 mV), and Lqh-II markedly increased the steady-state fast and slow inactivation. To mimic electrophysiological conditions we induced fast depolarization of toxin-bound synaptosomes, which generated a biphasic unbinding of Lqh-II from toxin-receptor complexes. The first fast off rate closely resembled values determined electrophysiologically for rBII in mammalian cells. The second off rate was similar to the voltage-independent steady-state value, attributed to binding to the slow-inactivated channel states. Thus, the Lqh-II voltage-dependent affinity highlights two independent mechanisms representing conformational changes of sodium channels associated with transitions among electrically visible and invisible inactivated states.
authors.affiliation: Commissaariat à l'Energie Atomique, Département d'Ingénierie et d'Etudes des Protéines, Centre d'Etudes de Saclay, 91191 Gif-sur-Yvette, France.
authors.forename: N
authors.initials: N
authors.lastname: Gilles
authors.forename: E
authors.initials: E
authors.lastname: Leipold
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: S H
authors.initials: SH
authors.lastname: Heinemann
authors.forename: D
authors.initials: D
authors.lastname: Gordon
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D008564:Membrane Potentials / Q000187:drug effects; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012604:Scorpion Venoms / Q000737:chemistry / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D013379:Substrate Specificity; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: C114938:Lqh II toxin; D009498:Neurotoxins; D012604:Scorpion Venoms; D015222:Sodium Channels