共查询到20条相似文献,搜索用时 31 毫秒
1.
Spider toxin (JSTX) on the glutamate synapse 总被引:1,自引:0,他引:1
A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate. 相似文献
2.
J Barhanin D Pauron A Lombet W Hanke G Boheim M Lazdunski 《Journal de physiologie》1984,79(4):304-308
Biochemical characterization of the Tityus gamma toxin receptor associated with the voltage-sensitive Na+ channel was carried out in different tissue preparations with the use of an iodinated toxin derivative. The affinity of the toxin for the receptor is high with a dissociation constant of 4 X 10(-12) M for rat synaptosomes. The density of binding sites is in the range of 0.3 to 2 pmol/mg of protein. Toxin gamma does not seem to bind to Na+ channels located on transverse-tubule membranes of skeletal muscle, but only to Na+ channels located on the sarcolemma. Both affinity labelling and radiation inactivation analysis indicate a molecular weight for the toxin receptor of 270 000 daltons. The same molecular weight is found using the tetrodotoxin. Only one single major protein component of the Na+ channel was purified from Electrophorus electroplax, rat brain membranes and chick heart membrane using the toxin gamma as a marker. The molecular weight of this component is 230 000-270 000 daltons. Reconstitution of the purified Na+ channel into planar lipid bilayers has been carried out. Two different types of electrically excitable channels with conductances of 150 and 25 pS were detected. The activity of both channels is blocked by saxitoxin. 相似文献
3.
Studies on nicotinic acetylcholine receptors in mammalian brain. Interaction of solubilized protein with cholinergic ligands 总被引:1,自引:0,他引:1
Binding of alpha-bungarotoxin, labeled with 125I, has been studied in detergent extracts and affinity purified acetylcholine receptor from rat cerebral cortex. Binding to detergent extracts is saturable and appears to be due to one class of binding sites present at a level of 0.27 pmol/mg of protein. The association constant is 2 X 10(7) liters mol-1 . Competition with cholinergic ligands indicates that toxin binding to both detergent solubilized and affinity purified receptor retains its nicotinic nature. Values for the ligand concentrations required to produce 50% inhibition of extent and rate of toxin binding are presented. 相似文献
4.
Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein. 相似文献
5.
本文合成了一种腺苷亲和层析凝胶,并采用亲和层析法从牛脑细胞膜上分离出了几种膜上结合的腺苷结合蛋白质。这些蛋白质在SDS-PAGE电泳凝胶上为单一或主要的蛋白带,分子量分别为64kd,45kd,35kd。腺苷转运体抑制剂潘生丁和NBMPR对64kd蛋白与^3h-腺苷的结合抑制作用远强于腺苷受体的激动剂NECA和R-PIA;这表明64kd蛋白为牛脑细胞膜上结合的腺苷转运体。 相似文献
6.
Six peptide toxins (Magi 1-6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier delta-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I-labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1-5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhalphaIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K(i)s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K(i)=267 nM) and site 4 in rat brain synaptosomes (K(i)=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel. 相似文献
7.
Evidence for distinct sites coupled to high affinity omega-conotoxin receptors in rat brain synaptic plasma membrane vesicles 总被引:3,自引:0,他引:3
P Feigenbaum M L Garcia G J Kaczorowski 《Biochemical and biophysical research communications》1988,154(1):298-305
The neuronal Ca2+ channel blocker omega-conotoxin (GVIA) binds with very high affinity (Kd of 0.8 pM) to a single class of receptors in purified rat brain synaptic plasma membrane vesicles. Three types of agents have been found to modulate toxin binding. The affinity of omega-conotoxin is decreased by metal ions or organic cations which interact at the pore of voltage-dependent Ca2+ channels. Dynorphin A [1-13] and related peptides stimulate omega-conotoxin binding by increasing toxin affinity through a nonopiate allosteric mechanism. Venom of the spider Plectreurys tristes inhibits omega-conotoxin binding (IC50 of 30 ng protein/ml) by a noncompetitive allosteric mechanism. These results suggest that omega-conotoxin binding sites exist in a complex with distinct receptors for other agents, all of which may be functionally associated with neuronal Ca2+ channels. 相似文献
8.
A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP. 相似文献
9.
The rat liver insulin receptor 总被引:1,自引:0,他引:1
K Hofmann H Romovacek G Titus K Ridge J A Raffensperger F M Finn 《Biochemistry》1987,26(23):7384-7390
Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Biochemical characterization of two nicotinic receptors from the optic lobe of the chick 总被引:4,自引:0,他引:4
We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor. 相似文献
11.
cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. 总被引:93,自引:0,他引:93
Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor. 相似文献
12.
The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein 总被引:4,自引:0,他引:4
S E Senogles J L Benovic N Amlaiky C Unson G Milligan R Vinitsky A M Spiegel M G Caron 《The Journal of biological chemistry》1987,262(10):4860-4867
Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary. 相似文献
13.
Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000. 相似文献
14.
Electrophysiological characterization, solubilization and purification of the Tityus gamma toxin receptor associated with the gating component of the Na+ channel from rat brain 总被引:5,自引:1,他引:4
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J Barhanin D Pauron A Lombet R I Norman H P Vijverberg J R Giglio M Lazdunski 《The EMBO journal》1983,2(6):915-920
Electrophysiological studies with neuroblastoma cells have shown that toxin gamma from the venom of the scorpion Tityus serrulatus is a new toxin specific for the gating system of the Na+ channel. The procedure which solubilizes the tetrodotoxin receptor from rat brain also solubilizes the Tityus gamma toxin receptor. Binding experiments on the solubilized receptor with a radioiodinated derivative of Tityus gamma toxin have shown: (i) that the TiTx gamma-receptor complex is very stable with a dissociation constant of 8.6 X 10(-12) M and a very slow dissociation (T 1/2 = 15 h); (ii) that the toxin recognizes a class of sites with a 1:1 stoichiometry with those for tetrodotoxin (Bmax = 1.3 pmol/mg protein). The radioiodinated Tityus gamma-receptor complex has been substantially purified by ion-exchange chromatography, lectin affinity chromatography and sucrose gradient sedimentation. A ratio of one Tityus gamma toxin binding site per tetrodotoxin binding site was found throughout the purification. The purified material exhibited a sedimentation coefficient of 10.4S and had an apparent mol. wt. of 270 000 on SDS-gel electrophoresis. No other polypeptide chains were demonstrated to be associated with this large protein in the Tityus gamma receptor. The main conclusion is that the tetrodotoxin binding site associated with the selectivity filter of the Na+ channel and the Tityus gamma toxin binding site associated with the gating component are probably carried by the same polypeptide chain. 相似文献
15.
The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified Gi, measured with GTPase and phospholipase C assays 总被引:3,自引:0,他引:3
H Ueda Y Yoshihara H Misawa N Fukushima T Katada M Ui H Takagi M Satoh 《The Journal of biological chemistry》1989,264(7):3732-3741
We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [3H]kyotorphin in rat brain membranes was composed of high affinity (Kd = 0.34 nM) and low affinity (Kd = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl2, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low Km GTPase activity. Kyotorphin produced a stimulation of low Km GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [3H]kyotorphin binding, yet in itself had no effect on the low Km GTPase. The kyotorphin stimulation of low Km GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, Gi, but not with Go. Similar evidence of selective coupling of kyotorphin receptor to Gi was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with Gi but not with Go. These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through Gi. This study provides the first evidence of a selective involvement of Gi in the receptor-mediated activation of phospholipase C. 相似文献
16.
Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit 总被引:3,自引:0,他引:3
The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit. 相似文献
17.
N. I. Kiskin E. M. Klyuchko O. A. Kryshtal' A. Ya. Tsyndrenko N. Akaike N. Kawai 《Neurophysiology》1989,21(2):110-116
The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10–4–10–5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 152–160, March–April, 1989. 相似文献
18.
A protein capable of binding atropine and (3H)propylbenzilylcholine mustard was solubilized and purified (200-fold) from rat brain. Pronase and trypsin, but not phospholipases, diminished the binding capacity of the solubilized receptor. The molecular weight of the salt-solubilized receptor as determined by gel filtration in the absence of detergents is 30,000. The purified protein showed specificity of binding toward muscarinic ligands. the high and low affinity dissociation constants of the receptor.atropine complex are 0.3 nM and 0.15 muM. Binding of atropine is pH-dependent with an optimum at 7.1. Ca2+ influences the binding of atropine and maximal binding occurs at 0.5 mM Ca2+. The subcellular distribution of the receptor was also examined. 相似文献
19.
Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation. 相似文献
20.
The spider toxin omega-agatoxin IIIA (omega-Aga-IIIA) is a potent inhibitor of high voltage-activated calcium currents in the mammalian brain. To establish the biochemical parameters governing its action, we radiolabeled the toxin and examined its binding to native and recombinant calcium channels. In experiments with purified rat synaptosomal membranes, both kinetic and equilibrium data demonstrate one-to-one binding of omega-Aga-IIIA to a single population of high affinity sites, with K(d) = approximately 9 pm and B(max) = approximately 1.4 pmol/mg protein. Partial inhibition of omega-Aga-IIIA binding by omega-conotoxins GVIA, MVIIA, and MVIIC identifies N and P/Q channels as components of this population. omega-Aga-IIIA binds to recombinant alpha(1B) and alpha(1E) calcium channels with a similar high affinity (K(d) = approximately 5-9 pm) in apparent one-to-one fashion. Results from recombinant alpha(1B) binding experiments demonstrate virtually identical B(max) values for omega-Aga-IIIA and omega-conotoxin MVIIA, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that omega-Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels. 相似文献