Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative potassium channel subunits and target size analysis

Seagar, Michael; Labbé-Jullié, Catherine; Granier, Claude; Göll, A.; Glossmann, Hartmut; Rietschoten, J. Van; Couraud, François

doi:10.1021/bi00362a010

Cited by 52 publications

(34 citation statements)

References 31 publications

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“…This polypeptide has also been covalently labeled in both intact cultured neurons and synaptic membranes from rat brain [9,10] and target size analysis suggests that this chain carries the binding site [lo]. A second polypeptide of 57-kDa was detected in liver but not in smooth or cardiac muscle.…”

Section: Resultsmentioning

confidence: 99%

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

1987

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High-affinity binding sites for m~no ['~~I]iodoapamin were detected in membranes (Kd = 59 pM, B,,, = 24 fmol/mg protein) and cultured cells (& = 69 pM, B,,, = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (& = 67 pM, B, , , = 42 fmol/mg protein) and liver (Kd = 15 pM, B,,, = 43 fmol/ mg protein). Binding was stimulated by K t ions = 0.3-0.5 mM). Covalent labeling with arylazide ['251]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85 -87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.Apamin, a bee-venom peptide, is a specific blocker of one type of CaZ +-activated K' channel. Although it was initially considered to be essentially a centrally acting neurotoxin, the first results demonstrating an effect of apamin in vitro were obtained in peripheral systems (for review see [l]) and its action on intestinal smooth muscle and hepatocyte suspensions has been well characterized using pharmacological and electrophysiological techniques [2 -71.Apamin can be radioiodinated to high specific activity and is a potentially useful ligand for a biochemical study of K + -channel-associated membrane binding sites. Work in this area has, however, been centered on the rat central nervous system receptor using covalent labeling, target-size analysis and detergent extraction and sedimentation analysis [8 -111. However, while high-affinity binding sites have also been described in guinea-pig but not rat hepatocytes [6] and in smooth muscle [7], no data on binding molecule structure in peripheral systems have, to our knowledge, been reported. In the present paper we describe photoaffinity labeling of the apamin receptor in plasma membrane fractions from guinea-pig ileum and liver. We also report the presence of apamin receptors in cultured rat cardiac cells and purified heart membranes. EXPERIMENTAL PROCEDURE Preparation of I-labeled 4-azido-2-nitrophenylaminoacetyl-apaminApamin was purified from bee venom [12], radioiodinated [ 131 and mono [' 251]iodo derivatives were separated by SP-(sulfopropy1)-Sephadex chromatography [14]. lz5I-1abeled 4-azido-2-nitrophenylaminoacetyl-apamin ('"I-ANPAAapamin) was prepared as previously described [9], in the presence of 1 mg/ml bovine serum albumin carrier protein, using a molar ratio of 4: 1 of azido-2-nitrophenylaminoacetyl- Membrane preparationSmooth muscle membranes were prepared from guineapig ileum by the method of Matlib et al. [l 51. After discontinuous density gradient centrifugation membranes from the 8 -30% sucrose interface were collected. In some experiments homogenization and preparation of the 100000 x g pellet were carried out in the presence of the protease inhibitors phenanthroline (1 mM) pepstatin A (1 pM), phenylmethylsulfonyl fluoride (0.1 mM) iodoacetamide (1 mM) and EDTA (2 mM) and the density-gradient step was omitted. Plasma membranes were prepared from guinea-pig liver following the procedure of Neville [16] up to step 11 and from ra...

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Section: Resultsmentioning

confidence: 99%

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

1987

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“…They served and disulphide-bond pattern, into seven groups, which generally reflect their natural origin : scorpion toxins of 31Ϫ39 amino as molecular calipers to measure the dimension of the outer vestibule of the pore of particular channels [40Ϫ47]. Furthermore, acids [9]; dendrotoxins from mamba snakes and kalicludines from sea anemone, of 57Ϫ60 amino acids [10Ϫ12] ; small pep-they were used for the purification, isolation [24, 48Ϫ50] and pharmacological classification of K ϩ channels [8], and to study tides of 18Ϫ22 amino acids isolated from bee venom, including mast-cell-degranulating peptide and apamin [13] ; sea anemone the tissue distribution [51,52] and physiological role [4,5] of individual channel subtypes. Since each K-toxin presents an intoxins of 35Ϫ37 amino acids [14]; the complex group of phospholipase A 2 toxins [15] from snake venoms, including crotoxin, dividual pattern of selectivity and specificity towards the vast array of existing K ϩ channels, the discovery of new toxins may notexin and beta-bungarotoxin [16] ; the hanatoxins of 35 amino acids isolated from spider venom [17]; and κ-conotoxin PVIIA, provide valuable tools for their further study.…”

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Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium‐channel‐blocking scorpion toxins

Selisko¹,

García²,

Becerril³

et al. 1998

European Journal of Biochemistry

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Potassium-channel-blocking scorpion toxins (A-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other A-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of A-K-toxins with K i values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and K V 1.1 K ϩ channels with moderate affinities, with K d values of around 0.7 µM and 4.1 µM (Shaker) and 0.5 µM and 1.0 µM (K V1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.Keywords : A-K-toxin ; Centruroides noxius ; cobatoxin; potassium channel; scorpion toxin.Potassium channels form a large family of integral mem-surements and mutational analyses, that they block K ϩ channels from the external side, occluding the channel pore [19Ϫ22]. Disbrane proteins that are present in almost every living cell. They play a key physiological role by setting the resting potential and placement experiments on rat brain synaptosomes provide evidence that the receptor site is shared by mast-cell-degranulating shaping bioelectric signals [1,2] PVIIA [27]. Thus, despite these peptides presenting very different three-dimensional folds [14, 28Ϫ34], they seem to act on Neurotoxins that selectively and sensitively block potassium channels (K-toxins) form a structurally diverse group, from K ϩ channels in a similar way. The only exception described has been hanatoxin [35,36]. small peptides to rather complex proteins. Some of them have been shown to be valuable pharmacological tools to study the Scorpion K-toxins were successfully used to identify the pore-forming region of K ϩ channels [20,37], to clarify the strucmolecular structure, function and diversity of K ϩ channels [8,9]. They can be grouped, based on the similarity of their size tural subunit stoichiometry [38], and to identify the functional stoichiometry of inactivation of K ϩ channels [39]. They served and disulphide-bond pattern, into seven groups, which generally reflect their natural origin : scorpion toxins of 31Ϫ39 amino as molecular calipers to measure the dimension of the outer vestibule of the pore of particular channels [40Ϫ47]. Furthermore, acids [9]; dendrotoxins from mamba snakes and kalicludines from sea anemone, of 57Ϫ60 amino acids [10Ϫ12] ; small pep-they were used for the purification, isolation [24, 48Ϫ50] and pharmacological classification of K ϩ channels [8], and to study tides of 18Ϫ22 amino acids isolated from bee venom, including ma...

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“…Apamin passes the blood/brain barrier where its site of action is the central nervous system (Habermann and Cheng-Raude, 1975). Banks et al (1979) discovered recently that apamin acts as a specific blocker of a calcium-dependent potassium channel in guinea-pig taenia coli and in liver, implying that apamin receptors are also located in the periphery.Receptor characterization was first made possible by the availability of a pure '251-labelled derivative of apamin (Hugues et al, 1982a) and later by the availability of two wellcharacterized photosensitive probes of '251-labelled apamin (Seagar et al, 1985(Seagar et al, , 1986. Studies on various tissues revealed a unique receptor in the rat central nervous system (Seagar et al, 1986), guinea-pig smooth muscle, liver and rat heart (Marqu2ze et al, 1987), which was composed of several protein subunits.…”

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“…Receptor characterization was first made possible by the availability of a pure '251-labelled derivative of apamin (Hugues et al, 1982a) and later by the availability of two wellcharacterized photosensitive probes of '251-labelled apamin (Seagar et al, 1985(Seagar et al, , 1986. Studies on various tissues revealed a unique receptor in the rat central nervous system (Seagar et al, 1986), guinea-pig smooth muscle, liver and rat heart (Marqu2ze et al, 1987), which was composed of several protein subunits.…”

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Binding and toxicity of apamin

Labbé-Jullié

Granier

Alberício

et al. 1991

European Journal of Biochemistry

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The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln-17 and the side chains of Arg-13 and Arg-14 (besides the charge effects). The interaction of apamin with its receptor, a calcium-activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.Of all the components characterized in the venom of the bee (Apis mellifera mellifera), the peptide apamin is the most toxic to mammals (Habermann and Reiz, 1965). Apamin passes the blood/brain barrier where its site of action is the central nervous system (Habermann and Cheng-Raude, 1975). Banks et al. (1979) discovered recently that apamin acts as a specific blocker of a calcium-dependent potassium channel in guinea-pig taenia coli and in liver, implying that apamin receptors are also located in the periphery.Receptor characterization was first made possible by the availability of a pure '251-labelled derivative of apamin (Hugues et al., 1982a) and later by the availability of two wellcharacterized photosensitive probes of '251-labelled apamin (Seagar et al., 1985(Seagar et al., , 1986. Studies on various tissues revealed a unique receptor in the rat central nervous system (Seagar et al., 1986), guinea-pig smooth muscle, liver and rat heart (Marqu2ze et al., 1987), which was composed of several protein subunits.Apamin is a basic peptide with 18 amino acid residues (Haux et al., 1967;Shipolini et al., 1967) and two disulfide bridges (Callewaert et al., 1968; Fig. 1). Solid-phase synthesis of apamin has been performed (Van Rietschoten et al., 1975) which permits structure/activity studies to be carried out, based on chemical modification of the natural peptide and which are complemented by studies on synthetic analogues. The activities were compared by determining the median lethal Abbreviations. Acm4-apamin, tetraacetamidomethyl apamin; (cHxO2]apamin, apamin modified on its two arginine residues by cyclohexanedione; LDS0, median lethal dose; apamin(x-y), synthetic analogue of apamin composed of amino acid residues x-y. dose (LD50) after subcutaneous injection in mice and for some, by binding experiments performed on rat brain membranes. In summary, the following observations were made. (a) Chemical modification of the natural peptide at the two free amino groups o...

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Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative potassium channel subunits and target size analysis

Cited by 52 publications

References 31 publications

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium‐channel‐blocking scorpion toxins

Binding and toxicity of apamin

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