Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

Seagar, M J; Labbé-Jullié, Catherine; Granier, Claude; Rietschoten, J Van; Couraud, François

doi:10.1016/s0021-9258(18)89204-3

Cited by 31 publications

(10 citation statements)

References 24 publications

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“…The autoradiogram shown in lane 3 (125I-eANPAA-apamin) required an exposure 3-4 times longer than that in lane 7 (125I-aANPAA-apamin). For this reason cross-linking of the lower molecular weight chains in synaptic membranes was overlooked in our first series of experiments using the undefined reaction mixture (Seagar et al, 1985). The omission of /3-mercaptoethanol during denaturation did not modify the migration of any of the detected bands.…”

Section: Photolabeling Of Primary Cultured Neurons and Synapticmentioning

confidence: 93%

“…Cerebral hemispheres from 16-day Wistar rat embryos were dissociated and cultured in poly(L-lysine)-treated 60-mm dishes (Corning) as previously described (Seagar et al, 1984). Synaptic membranes were prepared as in Seagar et al (1985). The binding buffer for neuronal cultures contained 25 mM Hepes, 10 mM glucose, 140 mM NaCl, 5.4 mM KC1, 1.8 mM CaCl2, 0.8 mM MgS04, and 0.1% serum albumin adjusted to pH 7.5 with Tris base.…”

Section: Methodsmentioning

confidence: 99%

“…Cells and membranes were labeled by using photoreactive ligands and then processed, in the presence of the protease inhibitors phenanthroline (1 mM), pepstatin A (1 mM) phenylmethanesulfonyl fluoride (100 mM), iodoacetamide (1 mM), and EDTA (1 mM), for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5-15% gradient) and autoradiography as previously described (Seagar et al, 1985).…”

Section: Methodsmentioning

confidence: 99%

“…Target Size Analysis. Crude synaptosomal membranes (Seagar et al, 1985) from Wistar rat cerebral cortex were prepared. After resuspension (10-15 mg of protein/mL) in degassed, serum albumin free binding buffer, supplemented with 100 mM phenylmethanesulfonyl fluoride and 1 mM EDTA, they were frozen in screwtop polypropylene vials (Nunc) and stored prior to and after irradiation in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…We have detected high-affinity binding sites for mono-[125I]iodoapamin on primary cultured neurons and have correlated receptor occupancy to an inhibition of ion efflux 0006-2960/86/0425-4051S01.50/0 © 1986 American Chemical Society ( Seagar et al, 1984). To try to identify receptor-associated polypeptides, we first randomly derivatized 125I-apamin with a photolabile arylazide and demonstrated covalent incorporation into membrane proteins (Seagar et al, 1985). Interpretation of these observations was difficult as several specific radioactive bands were resolved, the labeling pattern in cultured neurons was different from that in synaptic membranes, and considerable discrepancy existed between our data and reports using a homobifunctional cross-linker (Schmid- Antomarchi et al, 1984).…”

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confidence: 99%

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

Seagar¹,

Labbé-Jullié²,

Granier³

et al. 1986

Biochemistry

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Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

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Section: Photolabeling Of Primary Cultured Neurons and Synapticmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Seagar¹,

Labbé-Jullié²,

Granier³

et al. 1986

Biochemistry

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Role of Asn² and Glu⁷ residues in the oxidative folding and on the conformation of the N‐terminal loop of apamin

et al. 2007

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Molecular properties of the apamin binding protein associated with a calcium activated potassium channel

Seagar¹,

Marqueze²,

Couraud³

1988

Pestic. Sci.

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Apamin, a 2‐kdalton peptide from bee venom, is a potent convulsant. Its neurotoxic action has been attributed to specific blockade, at nanomolar concentrations, of the slow Co2+ activated K+ current (IAHP) that underlies the late afterhyperpolarisation in certain neurons. Mono[125I] iodoapamin binds with high affinity to intact cultured neurons (Kd = 30 PM, Bmax = 500‐1000 sites/cell) and synoptic membranes (Kd = 30 PM, Bmax = 30 fmol mg protein−1). Apamin contains two primary amines (alpha cysl and epsilon lys4) that can be modified to produce photoactivatable arylazide derivatives. Photoaffinity labeling with these derivatives has identified receptor polypeptides of 86 and 59 kdaltons accessible from the alpha cysl position and 33 kdaltons accessible from the epsilon lys4 position of the bound ligand. This suggests that apamin binds at the interface between 3 putative K+ channel subunits. Radiation inactivation studies indicated a target size of 84‐115 kdaltons. The simplest interpret at ion is that the 86 kdalton chain alone carries the neurotoxin binding site. The apamin binding protein can be extracted from rat brain membranes using sodium cholate. Scatchard analysis of equilibrium binding data revealed a single class of non‐interacting sites with Kd = 40 pm and Bmax = 17 fmol mg protein−1. As in membrane‐inserted receptors, apamin binding is stimulated by occupation of a K+ ion site that saturates at low millimolar concentrations. Other cations can be substituted for K+ with an affinity sequence: K+ = Tl+ = Rb+ > Cs+ > NH+4 > Li+ or Na+. The K+ channel Mockers quinidine and tetraethylammonium + displaced apamin from its solubilised receptor at concentrations similar to those required to block flux through apamin‐sensitive channels. Analysis by sucrose gradient centrifugation showed that the receptor sediments with S20=20, corresponding to a molecular weight of about 700 kdaltons. However, as much as 50%of this could be contributed by the detergent. Receptor sites for [125I]‐apamin have also been detected and identified by photoaffinity labeling in primary cultured astrocytes and heart cells as well as in membrane preparations from liver and smooth muscle. A 86–87 kdalton componet was was present in all these tissues whereas the 57–59 and 33kdalton polypeptides were revealed in some but not in others, These observations support the hypothesis that the larger chain contains the apamin binding site while the two smaller polypeptides are noncovalently associated perpheral subunits.

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Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

Cited by 31 publications

References 24 publications

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

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

Role of Asn² and Glu⁷ residues in the oxidative folding and on the conformation of the N‐terminal loop of apamin

Molecular properties of the apamin binding protein associated with a calcium activated potassium channel

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Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

Cited by 31 publications

References 24 publications

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

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

Role of Asn2 and Glu7 residues in the oxidative folding and on the conformation of the N‐terminal loop of apamin

Molecular properties of the apamin binding protein associated with a calcium activated potassium channel

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Role of Asn² and Glu⁷ residues in the oxidative folding and on the conformation of the N‐terminal loop of apamin