Characterization of a Novel Radiolabeled Peptide Selective for a Subpopulation of Voltage-gated Potassium Channels in Mammalian Brain

Racapé, Judith; Lecoq, Alain; Romi-Lebrun, Régine; Liu, Jessica; Köhler, Martin; García, María L.; Ménèz, Andre; Gasparini, Sylvaine

doi:10.1074/jbc.m109886200

Cited by 21 publications

(31 citation statements)

References 31 publications

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“…Proteins-BgK and BgK(W5Y/Y26F), an analog that can be radiolabeled without loss of biological activity (8), were synthesized as previously described (8). Synthetic charybdotoxin (ChTX) 1 was purchased from Latoxan (Valence, France), and kaliotoxin (KTX) and ShK were from Bachem (Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…cDNAs encoding hKv1.1 and hKv1.3 were cloned into the mammalian expression vector pCI-neo (Promega). The cDNA encoding hKv1.2 in pGEMA was kindly provided by Prof. Stephan Grissmer (Department of Applied Physiology, University of Ulm, Germany) and subcloned into the mammalian expression vector pcDNA3.1/HisC (Invitrogen) as described in (8). Plasmids were amplified in Escherichia coli XL1Blue using the plasmid purification kit from Qiagen (maxi protocol).…”

Section: Methodsmentioning

confidence: 99%

“…Concentrations of BgK and ChTX were obtained by absorbance determination at 280 nm, whereas concentrations of KTX and ShK were assessed from amino acid composition analyses performed on an AminoTag (Jeol). BgK(W5Y/Y26F) was radiolabeled with 125 I as previously described (8). Heterologous Expression of Kv1 Channels in Mammalian Cells-TsA-201 cells were maintained in 10-cm-diameter tissue culture dishes as previously described (8).…”

Section: Methodsmentioning

confidence: 99%

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A Variable Residue in the Pore of Kv1 Channels Is Critical for the High Affinity of Blockers from Sea Anemones and Scorpions

Gilquin

Braud

Eriksson

et al. 2005

Journal of Biological Chemistry

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Animal toxins are associated with well defined selectivity profiles; however the molecular basis for this property is not understood. To address this issue we refined our previous three-dimensional models of the complex between the sea anemone toxin BgK and the S5-S6 region of Kv1.1 (Gilquin, B., Racape, J., Wrisch, A., Visan, V., Lecoq, A., Grissmer, S., Mé nez, A., and Gasparini, S. (2002) J. Biol. Chem. 277, 37406 -37413) using a docking procedure that scores and ranks the structures by comparing experimental and back-calculated values of coupling free energies ⌬⌬G int obtained from doublemutant cycles. These models further highlight the interaction between residue 379 of Kv1.1 and the conserved dyad tyrosine residue of BgK. Because the nature of the residue at position 379 varies from one channel subtype to another, we explored how these natural mutations influence the sensitivity of Kv1 channel subtypes to BgK using binding and electrophysiology experiments. We demonstrated that mutations at this single position indeed suffice to abolish or enhance the sensitivity of Kv1 channels for BgK and other sea anemone and scorpion toxins. Altogether, our data suggest that the residue at position 379 of Kv1 channels controls the affinity of a number of blocking toxins.Molecular recognition and specific association of protein ligands and protein targets are central to most biological processes. Understanding the molecular basis of these interactions is critical for engineering novel protein-protein interactions. In particular, understanding how protein ligands bind with high affinity to only a subset of closely related receptors may help to design ligands with novel selectivities.A number of studies have been carried out to identify the sites used by protein ligands to bind to several related receptors. These sites were shown to be composed of a core formed by conserved hot spot residues together with target-specific residues (1-5). However, how these sites accommodate the different receptors subtypes is still poorly understood. In many cases the molecular determinants responsible for protein ligand discrimination remain to be identified.Toxins from sea anemones and scorpions that block currents through Kv1 voltage-gated potassium channels are particularly appropriate to investigate the molecular basis of selectivity of protein-protein interactions since each toxin binds to only

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Variable Residue in the Pore of Kv1 Channels Is Critical for the High Affinity of Blockers from Sea Anemones and Scorpions

Gilquin

Braud

Eriksson

et al. 2005

Journal of Biological Chemistry

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“…c Model representing the active surface of BgK, ShK and APETx1. Coloured amino acids represent key residues for the binding and function of BgK (Gilquin et al 2002;Racape et al 2002), ShK (Rauer et al 1999), and APETx1 (Chagot et al 2005a;Zhang et al 2007). Hydrophobic, basic and aliphatic residues are highlighted in green, blue, and yellow, respectively C2-C4, C3-C5 (Fig.…”

Section: Sea Anemone K + Channel Toxin Structuresmentioning

confidence: 99%

“…Alanine mutation analyses have shown that other residues (I7, R11, H19, R24 and F27 in ShK), which are clustered around the three conserved residues, are also important for the binding activity of ShK to Kv1.2 channels in brain membranes, and to Kv1.3 channels in T lymphocytes (Pennington et al 1996a, b;Rauer et al 1999). For BgK, other residues such as F6 and N19 are also involved in the binding and selectivity to Kv1 channels (Racape et al 2002). Molecular models of complexes between sea anemone toxins and Kv1 channels have been developed using the structure of KcsA (Doyle et al 1998;Lanigan et al 2002).…”

Section: Sea Anemone Toxins Interacting With Kv1 Channelsmentioning

confidence: 99%

Developmental Biology of Neoplastic Growth

Macieira‐Coelho¹

2005

Progress in Molecular and Subcellular Biology

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Contribution of the functional dyad of animal toxins acting on voltage‐gated Kv1‐type channels

Mouhat

Waard

Sabatier

2004

Journal of Peptide Science

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The 'functional dyad', a well-defined pair of amino acid residues (basic and hydrophobic residues), is a key molecular determinant present in most animal toxins acting on voltage-gated Kv1 channels. It is increasingly used as a working concept to explain how toxins are able to recognize and block their specific ion channel targets. However, other crucial toxin determinants are emerging and the actual role of this 'functional dyad' ought to be clarified, which is the object of the present mini-review.

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Characterization of a Novel Radiolabeled Peptide Selective for a Subpopulation of Voltage-gated Potassium Channels in Mammalian Brain

Cited by 21 publications

References 31 publications

A Variable Residue in the Pore of Kv1 Channels Is Critical for the High Affinity of Blockers from Sea Anemones and Scorpions

A Variable Residue in the Pore of Kv1 Channels Is Critical for the High Affinity of Blockers from Sea Anemones and Scorpions

Developmental Biology of Neoplastic Growth

Contribution of the functional dyad of animal toxins acting on voltage‐gated Kv1‐type channels

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