The Journal of Neuroscience, July 2, 2008, 28(27):6884-6894; doi:10.1523/JNEUROSCI.1228-08.2008
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Cellular/Molecular
Non-Agonist-Binding Subunit Interfaces Confer Distinct Functional Signatures to the Alternate Stoichiometries of the 
4β2 Nicotinic Receptor: An 4–4 Interface Is Required for Zn2+ Potentiation
Mirko Moroni,1
Ranjit Vijayan,2,3
Anna Carbone,1
Ruud Zwart,4
Philip C. Biggin,2 and
Isabel Bermudez1
1School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom, 2Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, 3Life Sciences Interface Doctoral Training Centre, University of Oxford, Oxford OX1 3QD, United Kingdom, and 4Lilly Research Centre, Windlesham, Surrey GU20 6PH, United Kingdom
Correspondence should be addressed to Isabel Bermudez, Gipsy Lane, Oxford OX3 0BP, UK. Email: p0054922{at}brookes.ac.uk
The 
4β2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The 4 and β2 nAChR subunits assemble into two alternate stoichiometries, (4)2(β2)3 and (4)3(β2)2, which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn2+. We show that Zn2+ exerts an inhibitory modulatory effect on (4)2(β2)3 receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (4)3(β2)2 receptors. Furthermore, Zn2+ inhibition on (4)2(β2)3 nAChRs is voltage-dependent, whereas it is not on (4)3(β2)2 receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn2+. Zn2+ inhibition is mediated by a site located on the β2(+)/4(–) subunit interfaces on both receptor stoichiometries. 4H195 and β2D218 are key determinants of this site. Zn2+ potentiation on (4)3(β2)2 nAChRs is exerted by a site that resides on the 4(+)/4(–) of this receptor stoichiometry. 4H195 on the (–) side of the ACh-binding 4 subunit and 4E224 on the (+) side of the non-ACh-binding 4 subunit critically contribute to this site. We also identified residues within the β2 subunit that confer voltage dependency to Zn2+ inhibition on (4)2(β2)3, but not on (4)3(β2)2 nAChRs.
Key words: Zn2+ modulation; nicotinic receptors; 4β2 receptors; subunit interfaces; acetylcholine; ACh
Received Nov. 19, 2007;
revised May 9, 2008;
accepted May 12, 2008.
Correspondence should be addressed to Isabel Bermudez, Gipsy Lane, Oxford OX3 0BP, UK. Email: p0054922{at}brookes.ac.uk
