Interactions between α-conotoxin MI and the Torpedo marmorata receptor α–δ interface

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Abstract

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the α–γ and α–δ subunit interfaces; α-conotoxins can bind them selectively. Moreover, we previously reported that α-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791–795]. Herein, to identify T. marmorata receptor regions involved in α-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. α-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the α/δ interface. A proposal for receptor–toxin interaction is discussed based on experimental results and docking studies.

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Materials and methods

Soybean l-α-phosphatidylcholine, carbamylcholine, Chloramine-T, N-hydroxysuccinimidyl-4-azido salicylic acid, and V8 proteinase (EC 3.4.21.19) were from Sigma Chemical Co., Saint Louis, MO, USA; modified trypsin (EC 3.4.21.4), was from Promega, Madison, WI, USA; Affi-Gel 10 from Bio-Rad, Richmond, CA, USA, and the carrier-free 125INa from NEN Research Products, New England Nuclear-DuPont Co., Boston, MA, USA. Bromoacetylcholine was synthesized from choline bromide and bromoacetyl bromide from

V8-Proteolytic digestion of the photoaffinity-labeled α and δ subunits

The complex nAChR-ASA-[125I]CnTx MI was submitted to the method of Pedersen and Cohen [5]. Gel bands were electroblotted onto a PVDF membrane, subjected to autoradiography and then sequenced. The autoradiography detected 4 and 3 labeled fragments from modified α and δ subunits, respectively (Fig. 1). Table 1 summarizes N-terminal sequences. The position of the low Mr radiolabeled band in Fig. 1B is coincident with that of the tracking dye. This band also contains a peptide mixture.

Localization of V8 labeled fragments

It was

Acknowledgments

Grants from CONICET and University of Buenos Aires supported this work.

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  • Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors

    2009, Biochemical Pharmacology
    Citation Excerpt :

    In earlier works the preparation and characterization of the peptide photoactivatable analogs in labeling of the whole subunits was achieved that first revealed the position of α-conotoxin-binding sites at the interfaces of the adjacent α1- and non-α1 nAChR subunits, as well as the involvement of the peptide N- and C-termini in the formation of complex with nAChR [76,77]. Later the employment of the phoactivatable analogs of α-conotoxins GI and MI resulted in detection of the crosslinked fragments of α1-, γ- and δ-subunits and partial mapping of the α1/γ[78] and α1/δ[79] sites from two Torpedo nAChRs, respectively. It is worth to mention, that goals of α-conotoxin analogs preparation are not limited to structural studies of nAChRs, but embrace numerous tasks (a fairly full list of synthesized α-conotoxin variants and derivatives is given in recent review [80]).

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