Bee Venom Neurotoxin (Apamin): Iodine Labeling and Characterization of Binding Sites

Abstract

1. Apamin, the octadecapeptide from bee venom acting on the central nervous system, has been labeled with lz5I at its single histidine residue. Apamin, monoiodoapamin, diiodoapamin, and iodide can be separated on thin-layer chromatography. Diiodoapamin retains about half the toxicity of the original peptide. Partial iodination with leading mainly to the monoiodo derivative, did not measurably decrease the toxicity.2. Labeled apamin binds to the insoluble, but not to the soluble part of homogenates made from rat cerebral cortex. The order of binding to homogenates is rat forebrain > rostra1 part of the brain stem > cerebellum > caudal part of the brain stem > spinal cord. Grey substance of rabbit forebrain binds much better than white matter. The binding site is enriched within fractions of rat cerebral cortex which sediment between 10000 and 100000 xg. 3. Apamin also binds to peripheral organs. In rats, the sequence of specific apamin binding by different organs is cerebral cortex % (20x) liver > kidney or ileum whereas binding by skeletal muscle, salivary glands, and testes is hardly measurable. In rabbits, the sequence is cerebral cortex NN liver > adrenals > cerebellum > kidney > medulla oblongata, whereas skeletal muscle and ileal mucosa do not bind to a measurable degree. Among all organs tested, bovine adrenal cortex binds the most. 4. Binding data with rat cerebral cortex were obtained under a variety of conditions. A single type of binding sites occurred with a density of about 1-3 pmol/g rat forebrain (wet weight). A very high affinity (Kd in the range of 10-25 pM) was found in both equilibrium and velocity measurements.5. With increasing temperature apamin binding is diminished. Labeled apamin is slowly degraded by the supernatant of rat brain homogenate at 37 "C and 25 "C, but not at 5 "C.High specificity, high affinity and prevalence of binding sites within the nervous system indicate, together with the rank order of potency of apamin derivatives, a role of binding in poisoning. However, final proof will need a direct comparison between binding and the action of apamin on isolated systems.Apamin is a specific, centrally acting neurotoxic peptide from bee venom [l]. Its structure has been determined independently by two groups [2,3] and solid-phase syntheses of fully active apamin have been achieved in two laboratories [4,5].Apamin hardly enters the brain or spinal cord upon intravenous injection [6]. Accordingly, apamin administered by intraventricular injection or microinjection into the spinal cord is 1000-10000 times more toxic than by intravenous or subcutaneous injection. In mice and rats about 10 ng of apamin, given intraventricularly, elicit the typical signs of taining 0.85 % sodium chloride.Abbreviation. Tris-saline, 0.05 M Tris-HCI buffer pH 7.5, contoxicity: after a short period of slightly depressed activity, the animals suffer from uncoordinated and generalized motor hyperactivity which eventually leads to death. Animals which survive sublethal doses show signs of hyperactivity and/or hy...

Role of small conductance calcium-activated potassium channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats

“…This may be due to apamin binds to a specific receptor that could result in protein pore conformation changes and subsequent phosphorylation, accounting for the observed steady state of apamin throughout the 3-h experimental period. The half-life of apamin is ϳ2 h, which is fairly consistent with results showing that apamin elicited relatively long-lasting response in the present study (19). It is well known that phosphorylation of K ϩ channels can alter neuronal excitability, and several SK channel residues have been identified as being susceptible to kinase phosphorylation, resulting in modulation of channel kinetics and changes in cellular localizations (1,42).…”

“…The relatively slower onset of apamin can be attributed to the complex three-dimensional structure of the peptide and correct conformational approach needed to bind to the apamin receptor compared with the smaller molecular weight of UCL1684 (27). The above hypothesis is supported by a radiolabeled study of apamin binding times (the half-life time of binding for apamin is ϳ31 min) (19). For UCL1684, in silico conformational studies have suggested a "cis" (synperiplanar) conformation as the likely active form of UCL1684, essentially acting as a cap over the outer pore (6).…”

Apamin as a selective blocker of the calcium-dependent potassium channel in neuroblastoma cells: voltage-clamp and biochemical characterization of the toxin receptor.

“…Ionic conditions are of particular importance for the binding of apamin to its receptor (14). Various monovalent and divalent cations prevent 125I-apamin binding to its receptor (Na+, guanidinium, choline, Ca +).…”

“…There is no tyrosine residue in the sequence, but there is histidine residue that is not essential for activity (4) and that can be iodinated. It has been found that the p9rocedure previously described to prepare "2I-labeled apamin (' I-apamin) (14) could not be used for the characterization of the '"I-apamin receptor on neuroblastoma cells. The labeled apamin obtained by this technique has a specific radioactivity of only 200-500 Ci/ mmol (1 Ci = 3.7 x 1010 becquerels).…”

Discrimination between subtypes of apamin‐sensitive Ca2+‐ activated K+ channels by gallamine and a novel bis‐quaternary quinolinium cyclophane, UCL 1530

“…We think this unlikely for several reasons. In the first place, the structurally related compound, dequalinium, has previously been shown not to block voltage-gated Ca2" channels in sympathetic neurones at concentrations up to 10 gM (Dunn, 1994 (Habermann & Fischer, 1979;Hugues et al, 1982;Habermann, 1984) and more recent work has shown that intracellular anions also affect the activity of a number of K+ channels, as well as their sensitivity to blocking agents (Zhang et al, 1994;Heath & Terrar, 1994;McKillen et al, 1994 (Wadsworth et al, 1994). This evidence is clearly consistent with the existence of SKCa channel subtypes.…”