Tài liệu Báo cáo khoa học: Activation of the Torpedo nicotinic acetylcholine receptor The

Activation of the Torpedo nicotinic acetylcholine receptor

The contribution of residues aArg55 and cGlu93

Ankur Kapur, Martin Davies, William F. Dryden and Susan M.J. Dunn

Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada

The muscle-type nicotinic acetylcholine receptor

(nAChR) is the prototype of the Cys-loop ligand-gated

ion channel (LGIC) super-family that includes the

neuronal nicotinic, c-aminobutyric acid (GABA) type

A, 5-hydrotryptamine type 3 (5-HT3) and glycine

receptors. This is largely a consequence of the abun￾dance of this receptor in Torpedo electric organ, which

facilitated its early purification and characterization.

The Torpedo nAChR is a pentameric transmembrane

protein complex in which four structurally related

subunits (a, b, c, d) in a stoichiometry of 2 : 1 : 1 : 1

assemble to form a central cation-selective ion channel

[1,2]. The a and b subunits of the Torpedo receptor

referred to in this report correspond to the a1 and b1

subunits in the nomenclature recommended by the

International Union of Pharmacology [3]. Radioligand

binding studies have demonstrated that, under equilib￾rium conditions, the nAChR carries two high affinity

Keywords

acetylcholine; loop D; mutagenesis; nicotinic

receptor; oocytes

Correspondence

S.M.J. Dunn, Department of Pharmacology,

University of Alberta, Edmonton, Alberta,

T6G 2H7 Canada

Fax: +780 4924325

Tel: +780 4923414

E-mail: [email protected]

(Received 22 October 2005, revised 13

December 2005, accepted 23 December

2005)

doi:10.1111/j.1742-4658.2006.05121.x

The Torpedo nicotinic acetylcholine receptor is a heteropentamer (a2bcd) in

which structurally homologous subunits assemble to form a central ion

pore. Viewed from the synaptic cleft, the likely arrangement of these sub￾units is a–c–a–d–b lying in an anticlockwise orientation. High affinity bind￾ing sites for agonists and competitive antagonists have been localized to

the a–c and a–d subunit interfaces. We investigated the involvement of

amino acids lying at an adjacent interface (c–a) in receptor properties.

Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used

to investigate the consequences of mutating aArg55 and cGlu93, residues

that are conserved in most species of the peripheral nicotinic receptors.

Based on homology modeling, these residues are predicted to lie in close

proximity to one another and it has been suggested that they may form a

salt bridge in the receptor’s three-dimensional structure (Sine et al. 2002 J

Biol Chem 277, 29 210–29 223). Although substitution of aR55 by phenyl￾alanine or tryptophan resulted in approximately a six-fold increase in the

EC50 value for acetylcholine activation, the charge reversal mutation

(aR55E) had no significant effect. In contrast, the replacement of cE93 by

an arginine conferred an eight-fold increase in the potency for acetyl￾choline-induced receptor activation. In the receptor carrying the double

mutations, aR55E-cE93R or aR55F-cE93R, the potency for acetylcholine

activation was partially restored to that of the wild-type. The results sug￾gest that, although individually these residues influence receptor activation,

direct interactions between them are unlikely to play a major role in the

stabilization of different conformational states of the receptor.

Abbreviations

5-HT3A receptor, serotonin type 3 A receptor; a-BgTx, alpha-bungarotoxin; ACh, acetylcholine; AChBP, acetylcholine binding protein;

dTC, d-tubocurarine; GABA, c-aminobutyric acid; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; PTMA,

phenyltrimethylammonium; WT, wild-type.

960 FEBS Journal 273 (2006) 960–970 ª 2006 The Authors Journal compilation ª 2006 FEBS