Variations in receptor site-3 on rat brain and insect sodium channels highlighted by binding of a funnel-web spider δ-atracotoxin

δ-Atracotoxins (δ-ACTXs) from Australian funnel-web spiders differ structurally from scorpion α-toxins (ScαTx) but similarly slow sodium current inactivation and compete for their binding to sodium channels at receptor site-3. Characterization of the binding of ¹²⁵I-labelled δ-ACTX-Hv1a to various sodium channels reveals a decrease in affinity for depolarized (0 mV; K_d = 6.5 ± 1.4 nM) vs.polarized (-55 mV; K_d = 0.6 ± 0.2 nM) rat brain synaptosomes. The increased K_d under depolarized conditions correlates with a 4.3-fold reduction in the association rate and a 1.8-increase in the dissociation rate. In comparison, ScαTx binding affinity decreased 33-fold under depolarized conditions due to a 48-fold reduction in the association rate. The binding of ¹²⁵I-labelled δ-ACTX-Hv1a to rat brain synaptosomes is inhibited competitively by classical ScαTxs and allosterically by brevetoxin-1, similar to ScαTx binding. However, in contrast with classical ScαTxs, ¹²⁵I-labelled δ-ACTX-Hv1a binds with high affinity to cockroach Na⁺ channels (K_d = 0.42 ± 0.1 nM) and is displaced by the ScαTx, LqhαIT, a well-defined ligand of insect sodium channel receptor site-3. However, δ-ACTX-Hv1a exhibits a surprisingly low binding affinity to locust sodium channels. Thus, unlike ScαTxs, which are capable of differentiating between mammalian and insect sodium channels, δ-ACTXs differentiate between various insect sodium channels but bind with similar high affinity to rat brain and cockroach channels. Structural comparison of δ-ACTX-Hv1a to ScαTxs suggests a similar putative bioactive surface but a 'slimmer' overall shape of the spider toxin. A slimmer shape may ease the interaction with the cockroach and mammalian receptor site-3 and facilitate its association with different conformations of the rat brain receptor, correlated with closed/open and slow-inactivated channel states.