Localization of receptor sites for insect-selective toxins on sodium channels by site-directed antibodies

Abstract

Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.

Binding of an α scorpion toxin to insect sodium channels is not dependent on membrane potential

“…The data presented in Fig. 1 reveal that the binding of LqhaIT to locust neuronal membranes was not affected by: (a) the excitatory and depressant insect selective scorpion toxins, shown to bind with high affinity to insect sodium channels [9,19,23]; (b) the /I toxin Ts VII, shown to compete with other /I toxins on binding to receptor site 4 in vertebrate sodium channels [24,25] as well as with the above insect-selective toxins on binding to insect sodium channels [19,17,26]; (c) TTX, the universal sodium blocker, binding to receptor site 1 in vertebrate [ 1,2] and insect [ 11,231 sodium channels and (d) the a scorpion toxin AaH II, extremely toxic to mammals, that binds to receptor site 3 in vertebrate sodium channels [ 1,2]. The latter is in accordance with previous results indicating that AaH II was not toxic to insects [13] and devoid of specific binding to insect neuronal membranes [ 193. On the other hand, sea anemone toxin ATX II, shown to competitively inhibit the binding of a scorpion toxin to receptor site 3 in vertebrate sodium channel [2], completely inhibits, with high affinity, ["'I]LqhaIT binding to insect neuronal membranes (Fig.…”

“…Co. Rockland, USA) according to the method described by [17], using 0.5 mCi carrier free ["'I]Na (Nuclear Research Center, Negev, Israel) and 5 pg of LqhaIT. The monoiodotoxin was purified as described [9] by a LiChrospher 100 RP-8 (5 pm) column (Merck). The concentration of the radiolabeled toxin was determined according to the specific radioactivity of the 12'1 corresponding to 2,424 dpm/fmol monoiodotoxin.…”

“…These were performed in the form of equilibrium saturation assays using increasing concentrations of the unlabeled toxin in the presence of a constant low concentration of the labeled toxin (0.1 l-0.2 nM), as described [9]. Analysis of the binding assays was performed using the iterative computer program LIGAND (P.J.…”

“…Termination, filtration (GFIF filters, Whatman, UK) and washing was according to [9]. Non-specific binding was determined in the presence of 1 PM unlabeled toxin and corresponded to 15525% of the total binding.…”

“…cently demonstrated that the two groups of insect-selective toxins bind to distinct receptor sites in insect sodium channels ( [9] and Moskowitz et al, unpublished).…”