Effect of Depolarization on Bindig Kinetics of Scorpion #alpha#-Toxin Highlights Conformational Changes of Rat Brain Sodium Channels

摘要

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 Lph-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 330fold decrease in toxin affinity for depolarized (0mV, 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. Elecrophysiological 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.