Scorpion Toxin Binding Research Articles

Abstract 2237 Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals sub-conductance that is abolished by PKA phosphorylation

Abstract 2237 Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals sub-conductance that is abolished by PKA phosphorylation

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.

Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1.

Chlorotoxin (ClTx) is a 36-residue disulfide-rich peptide isolated from the venom of the scorpion Leiurus quinquestriatus. This peptide has been shown to selectively bind to brain tumours (gliomas), however, with conflicting reports regarding its direct cellular target. Recently, the vascular endothelial growth factor receptor, neuropilin-1 (NRP1) has emerged as a potential target of the peptide. Here, we sought to characterize the details of the binding of ClTx to the b1-domain of NRP1 (NRP1-b1) using solution state nuclear magnetic resonance (NMR) spectroscopy. The 3D structure of the isotope labelled peptide was solved using multidimensional heteronuclear NMR spectroscopy to produce a well-resolved structural ensemble. The structure points to three putative protein-protein interaction interfaces, two basic patches (R14/K15/K23 and R25/K27/R36) and a hydrophobic patch (F6/T7/T8/H10). The NRP1-b1 binding interface of ClTx was elucidated using 15N chemical shift mapping and included the R25/K27/R36 region of the peptide. The thermodynamics of binding was determined using isothermal titration calorimetry (ITC). In both NMR and ITC measurements, the binding was shown to be competitive with a known NRP1-b1 inhibitor. Finally, combining all of this data we generate a model of the ClTx:NRP1-b1 complex. The data shows that the peptide binds to the same region of NRP1 that is used by the SARS-CoV-2 virus for cell entry, however, via a non-canonical binding mode. Our results provide evidence for a non-standard NRP1 binding motif, while also providing a basis for further engineering of ClTx to generate peptides with improved NRP1 binding for future biomedical applications.

Induced-Fit Pathway Accelerated Binding of Agitoxin-2 to A K+ Channel Imaged by HS-AFM

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels which consists of 38 amino acids. The docking model has been elucidated by using solution structure of AgTx2 and molecular dynamics simulation with K+ channel, thus electrostatic and hydrophobic interactions that contribute to binding have been revealed. However, it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

Open conformation of hERG channel turrets revealed by a specific scorpion toxin BmKKx2.

BackgroundThe human ether-a-go-go-related gene potassium channel (hERG) has an unusual long turret, whose role in recognizing scorpion toxins remains controversial. Here, BmKKx2, the first specific blocker of hERG channel derived from scorpion Mesobuthus martensii, was identified and the turret role of hERG channel was re-investigated using BmKKx2 as a molecular probe.ResultsBmKKx2 was found to block hERG channel with an IC50 of 6.7 ± 1.7 nM and share similar functional surface with the known hERG channel inhibitor BeKm-1. The alanine-scanning mutagenesis data indicate that different residue substitutions on hERG channel by alanine decreased the affinities of toxin BmKKx2 by about 10-fold compared with that of wild-type hERG channel, which reveals that channel turrets play a secondary role in toxin binding. Different from channel turret, the pore region of hERG channel was found to exert the conserved and essential function for toxin binding because the mutant hERG-S631A channel remarkably decreased toxin BmKKx2 affinity by about 104-fold.ConclusionsOur results not only revealed that channel turrets of hERG channel formed an open conformation in scorpion toxin binding, but also enriched the diversity of structure-function relationships among the different potassium channel turrets.

The Molecular Mechanism of Toxin-Induced Conformational Changes in a Potassium Channel: Relation to C-Type Inactivation

Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.

A new class of scorpion toxin binding sites related to an A-type K + channel: pharmacological characterization and localization in rat brain

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensi venom, sequenced and chemically synthesized (sBmTX3). The A-type current of striatum neurons in culture completely disappeared when 1 μM sBmTX3 was applied ( K d=54 nM), whereas the sustained K + current was unaffected. 125I-sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol mg −1 of protein, K d=0.21 nM). A panel of toxins yet described as specific ligands for K + channels were unable to compete with 125I-sBmTX3. A high density of 125I-sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Molecular modeling of scorpion toxin binding to voltage-gated K+ channels

Mutational studies have identified part of the S5-S6 loop of voltage-dependent K+ channels (P region) responsible for tetraethylammonium (TEA) block and permeation properties. Several scorpion peptide toxins – charybdotoxin (ChTX), kaliotoxin (KITX), and agitoxin (AgTX) – also block the channel with high affinity and specificity. An interaction surface for the toxins has been identified by mutation-induced alteration in toxin binding, and some of the interaction partners have been identified by mutation of channel residues using mutant cycle analysis. This has provided a general picture for the channel vestibule. Here, we examine the interaction predicted when the scorpion toxins are docked onto a molecular model of the K+ channel pore (the Kv1.3 isoform) that we recently proposed [1]. In the optimal alignment of the toxin with the pore, Arg-24 of KITX or AgTX forms a hydrogen bond with the Asp-386 carboxyl of one subunit, and Asn-30 is in immediate contact with Asp-386 of the opposing subunit in the channel tetramer. Toxin residues in proximity to the side chain of Lys-27 (Phe-25, Thr-36, Met-29, and Ser-11 in KITX) interact with the outer ring of four C-end His-404 residues. For ChTX the interaction with Asp-386 is reduced, but this is compensated by additional non-bonded interactions formed by Tyr-36 and Arg-34. Comparison of calculated energy of interaction of these specific toxin-channel residue pairs with experimental studies reveals good agreement. The similar total calculated energy of interaction is consistent with the similar IC50 for Kv1.3 block by KITX and ChTX. Molecular modeling shows complementarity of the pore model to toxin spatial structures, and contributes to our understanding of the surface topology to the K+ channel outer vestibule.

δ-Atracotoxins from Australian funnel-web spiders compete with scorpion α-toxin binding on both rat brain and insect sodium channels

δ-Atracotoxins are novel peptide toxins from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. To analyse their interaction with known sodium channel neurotoxin receptor sites we determined their effect on scorpion toxin, batrachotoxin and saxitoxin binding. Nanomolar concentrations of δ-atracotoxin-Hv1 and δ-atracotoxin-Ar1 completely inhibited the binding of the scorpion α-toxin AaH II to rat brain synaptosomes as well as the binding of LqhαIT, a scorpion α-toxin highly active on insects, to cockroach neuronal membranes. Moreover, δ-atracotoxin-Hv1 cooperatively enhanced batrachotoxin binding to rat brain synaptosomes in an analogous fashion to scorpion α-toxins. Thus the δ-atracotoxins represent a new class of toxins which bind to both mammalian and insect sodium channels at sites similar to, or partially overlapping with, the receptor binding sites of scorpion α-toxins.

A model of scorpion toxin binding to voltage-gated K+ channels.

Mutational studies have identified part of the S5-S6 loop of voltage-dependent K+ channels (P region) responsible for tetraethylammonium (TEA) block and permeation properties. Several scorpion peptide toxins-charybdotoxin (ChTX), kaliotoxin (KITX), and agitoxin (AgTX)-also block the channel with high affinity and specificity. Here, we examine the interaction predicted when the toxins are docked onto the molecular model of the K+ channel pore that we recently proposed. Docking with the model of the Kv1.3 channel started by location of Lys-27 side chain into the central axis of the pore, followed by energy minimization. In the optimal arrangement, Arg-24 of KITX or AgTX forms a hydrogen bond with the Asp-386 carboxyl of one subunit, and Asn-30 is in immediate contact with Asp-386 of the opposing subunit in the tetramer. Toxin residues in proximity to the side chain of Lys-27 (Phe-25, Thr-36, Met-29, and Ser-11 in KITX) interact with the four C-end His-404s. For ChTX the interaction with Asp-386 is reduced, but this is compensated by additional nonbonded interactions formed by Tyr-36 and Arg-34. Comparison of calculated energy of interaction of these specific toxin-channel residues with experimental studies reveals good agreement. The similar total calculated energy of interaction is consistent with the similar IC50 for Kv1.3 block by KITX and AgTX. Steric contacts of residues in position 380 of the S5-P linker with residues on the upper part of toxins permit reconstruction of the K+ channel outer vestibule walls, which are about 30 A apart and about 9 A high. Molecular modeling shows complementarity of the pore model to toxin spacial structures, and supports the proposal that the N-terminal borders of the P regions surround residues of their C-terminal halves.

An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula

The Kv2.1 voltage-activated K + channel, a Shab-related K + channel isolated from rat brain, is insensitive to previously identified peptide inhibitors. We have isolated two peptides from the venom of a Chilean tarantula, G. spatulata, that inhibit the Kv2.1 K + channel. The two peptides, hanatoxin, (HaTx 1) and hanatoxin 2 (HaTx 2), are unrelated in primary sequence to other K + channel inhibitors. The activity of HaTx was verified by synthesizing it in a bacterial expression system. The concentration dependence for both the degree of inhibition at equilibrium (K d = 42 nM) and the kinetics of inhibition (k on = 3.7 × 10 4 M −1s −1; k off = 1.3 × 10 −3 s −1), are consistent with a bimolecular reaction between HaTx and the Kv2.1 K + channel. Shaker-related, Shaw-related, and eag K + channels were relatively insensitive to HaTx, whereas a Shal-related K + channel was sensitive. Regions outside the scorpion toxin binding site (S5-S6 linker) determine sensitivity to HaTx. HaTx introduces a new class of K + channel inhibitors that will be useful probes for studying K + channel structure and function.

Localization of voltage-sensitive sodium channels on the extrasynaptic membrane surface of mouse skeletal muscle by autoradiography of scorpion toxin binding sites

Voltage-dependent sodium channels (Na+ channels) were localized by autoradiography on mouse skeletal muscle using both light and electron microscopy. 125I-scorpion toxins (ScTx) of both the alpha and beta type were used as probes. The specificity of labelling was verified by competitive inhibition with unlabelled toxin and by inhibition of alpha ScTx labelling in depolarizing conditions. Under light microscopy, the labelling of the myocyte surface appeared randomly distributed with both the alpha and beta toxins. No difference in the labelling density obtained with beta ScTx was observed between a 2 mm central segment of the fibre containing the endplate and an adjacent segment not containing the endplate. At the endplate, however, the beta ScTx binding site density was about seven fold higher at the edge of the synaptic primary clefts. This density decreased with distance from the synaptic cleft reaching the extrasynaptic value at 30-40 microns. An analysis of myocyte labelling using electron microscopy provided evidence for a specific, but very low labelling of the myocyte interior which can be attributed to the T-tubules. These results confirm a relatively high density of Na+ channels in a perijunctional zone about 50 microns in width, which could ensure the initial spread of the surface depolarization with a high safety factor, and a homogeneous distribution over the remaining surface with a low density evaluated at 5-10 per microns2. However, the very low labelling of T-tubules could be attributed mainly to a low density of tubular Na+ channels.

Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes.

A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.

Development of Sodium Channel Protein During Chemically Induced Differentiation of Neuroblastoma Cells

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

3H-batrachotoxinin-A benzoate binding to voltage-sensitive sodium channels: inhibition by the channel blockers tetrodotoxin and saxitoxin

The sodium channel blockers tetrodotoxin (TTX) and saxitoxin (STX) and the channel activator batrachotoxin (BTX) produce their effects by binding to separate and distinct sites on the channel protein. The fact that TTX- and STX-modified sodium channels are blocked to sodium flux has precluded drawing any direct conclusions regarding the effect of TTX/STX on BTX binding based on electrophysiological or 22Na flux measurements. Nevertheless, these sites have been presumed to be non-interacting. In this study, 3H-batrachotoxinin-A benzoate (BTX-B), a tritiated congener of BTX, has been used to provide a direct assessment of these binding interactions. Equilibrium specific binding of 3H-BTX-B to sodium channels in vesicular preparations of mouse brain in the presence of scorpion toxin was measured using a filtration assay procedure. At 25 degrees C both TTX and STX inhibit 3H-BTX-B binding in a concentration-dependent and noncompetitive manner. This inhibition is markedly temperature-dependent, being negligible at 37 degrees C and maximal at 18 degrees C, the lowest temperature investigated. Scatchard analysis of BTX-B binding isotherms at 25 degrees C in the presence and absence of 1 microM TTX revealed that inhibition is due to a 3-fold decrease in the affinity of BTX-B binding with no change in the number of binding sites (Bmax). The concentration dependence for TTX inhibition of both specific 3H-STX and 3H-BTX-B binding is identical, suggesting that inhibition of 3H-BTX-B binding is due to a direct effect of TTX/STX binding at their specific sodium channel site. The channel blockers did not alter the binding of scorpion toxin under these assay conditions, nor did BTX-B affect the binding of 3H-STX.(ABSTRACT TRUNCATED AT 250 WORDS)

The sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition.

Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated 22Na+ influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Binding of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which [Na+]out/[Na+]in is varied by changing [Na+]in or [Na+]out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.

Interaction of brevetoxin A with a new receptor site on the sodium channel

W. A. Catterall and M. Gainer. Interaction of brevetoxin A with a receptor site on the sodium channel. Toxicon 23, 497 – 504, 1985. — Measurements of neurotoxin-activated 22Na influx in neuroblastoma cells and neurotoxin binding in synaptosomes were used to define the site and mechanism of action of brevetoxins on sodium channels. Brevetoxin A alone did not enhance the sodium permeability of neuroblastoma cells, but markedly enhanced persistent activation of sodium channels by veratridine, aconitine and batrachotoxin, which act at neurotoxin receptor site 2. Enhancement of batrachotoxin action was accompanied by a 4.3-fold increase in the binding of [ 3H]batrachotoxinin A 20-α-benzoate to receptor site 2 on sodium channels in synaptosomes. These results point to an allosteric mechanism of brevetoxin action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels at receptor site 2. Brevetoxin A does not block [ 3H]saxitoxin binding at neurotoxin receptor site 1 or 125I-labeled scorpion toxin binding at neurotoxin receptor site 3. The brevetoxins appear to act at a new neurotoxin receptor site on the sodium channel.

Cyclic AMP-dependent phosphorylation of the alpha subunit of the sodium channel in synaptic nerve ending particles.

In purified preparations of voltage-sensitive sodium channels, the alpha subunit is selectively phosphorylated by cAMP-dependent protein kinase (Costa, M. R. C., Casnellie, J. E., and Catterall, W. A. (1982) J. Biol. Chem., 7918-7921). We have developed methods to measure sodium channel phosphorylation in both lysed synaptosomal membranes and intact synaptosomes. Incubation of lysed synaptosomal membranes with exogenously added catalytic subunit of cAMP-dependent kinase and [gamma-32P]ATP resulted in rapid phosphorylation of the alpha subunit as detected by specific immuno-precipitation, sodium dodecyl sulfate-gel electrophoresis, and autoradiography. Analysis of tryptic phosphopeptides revealed five major sites of reaction. The level of phosphorylation of these sites on the sodium channel in intact synaptosomes was monitored using a rephosphorylation method in which those sites not phosphorylated in situ were labeled with [gamma-32P]ATP and exogenously added protein kinase after lysis of the synaptosomes. Incubation of synaptosomes with 8-Br-cAMP completely blocked labeling of the alpha subunit in rephosphorylation indicating marked stimulation of phosphorylation of the sites on the sodium channel in situ. Phosphorylation was complete in 15 s and all four of the tryptic phosphopeptides detected under these conditions could be phosphorylated in situ. These results show that the sodium channel can be rapidly phosphorylated by endogenous cAMP-dependent protein kinase in intact synaptosomes. In addition, since ATP and protein kinase are only available inside the synaptosomes, they also show that the alpha subunit is a transmembrane polypeptide exposed on both sides of the synaptosomal membrane. The functional consequences of 8-Br-cAMP-stimulated phosphorylation were examined using ion flux and neurotoxin-binding methods. Binding of saxitoxin and scorpion toxin were unaffected, but neurotoxin-activated 22Na+ influx mediated by the sodium channel was reduced 16 to 26% (P less than 0.01) under various experimental conditions. The potential physiological significance of this action is considered.

The sodium channel from rat brain. Reconstitution of neurotoxin-activated ion flux and scorpion toxin binding from purified components.

The rat brain Na+ channel was purified to homogeneity and reconstituted into pure egg phosphatidylcholine vesicles or vesicles composed of a mixture of egg phosphatidylcholine and rat brain lipid. In each case, the binding affinities at 37 degrees C for saxitoxin (STX) and tetrodotoxin (TTX) are nearly identical with those measured for intact Na+ channels. Approximately 50% of the reconstituted channels are oriented right-side-out. Veratridine stimulates the initial rate of 22Na+ uptake 8- to 15-fold with a K0.5 of 28 microM. External TTX blocks the fraction of Na+ channels oriented right-side-out with a Ki of 14 nM. All of the veratridine-stimulated 22Na+ uptake is blocked when TTX is present on both sides of the vesicle membrane, or when tetracaine is added to the external medium. The veratridine-activated reconstituted Na+ channel transports cations with a permeability ratio of PNa+/PRb+/PCa+ = 1.0:0.25:0.12. We estimate that at least 30% and perhaps as many as 70% of the reconstituted channels are active. Purified sodium channels reconstituted in egg phosphatidylcholine vesicles do not bind 125I-scorpion toxin (125I-LqTx). In contrast, when incorporated into vesicles containing rat brain lipids, 76% of the Na+ channels bound 125I-LqTx with an average KD of 80 nM. Thermal denaturation of purified Na+ channels at 36 degrees C prior to reconstitution causes a parallel loss of both the [3H]STX- and 125I-LqTx-binding activity measured after reconstitution. Sea anemone toxin II displaces bound 125I-LqTx with a KD 60-fold greater than that of unlabeled LqTx. These data indicate that the alpha, beta 1, and beta 2 subunits of the sodium channel are sufficient for reconstitution of both selective, veratridine-stimulated ion transport and 125I-LqTx binding.

Developmental and neurochemical specificity of neuronal deficits produced by electrical impulse blockade in dissociated spinal cord cultures.

Blockade of spontaneous electrical activity in dissociated fetal spinal cord cultures produced neuronal deficits as measured by biochemical and morphological techniques. Spinal cord cultures exhibited an age-dependent vulnerability to impulse blockade with tetrodotoxin (TTX) or xylocaine. Neuronal cell counts, [125I]tetanus toxin fixation and [125I]scorpion toxin binding indicated that TTX application produced neuronal deficits during the second or third week in culture. Application of TTX during the first or fourth week did not produce a difference in tetanus toxin fixation from controls. Radioautography of [125I]tetanus toxin revealed no obvious change in the label distribution after TTX treatment. Suppression of electrical activity during the first 6 days in culture had no effect on choline acetyltransferase (CAT) activity and no apparent effect on the appearance of the cultures. Application of TTX during the seventh day in culture decreased CAT activity to 68% of control. Chronic electrical blockade produced a progressively greater loss of CAT activity through 21 days in culture. GABAergic neurons, as indicated by high-affinity GABA uptake, glutamic acid decarboxylase activity and [3H]GABA radioautography, were not affected by electrical blockade. These data indicate that there is developmental and neurochemical specificity in the neuronal death produced by blocking spontaneous electrical activity in dissociated spinal cord cultures.