Non-agonist-binding subunit interfaces confer distinct functional signatures to the alternate stoichiometries of the alpha4beta2 nicotinic receptor: an alpha4-alpha4 interface is required for Zn2+ potentiation.
The alpha4beta2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The alpha4 and beta2 nAChR subunits assemble into two alternate stoichiometries, (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(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 (alpha4)(2)(beta2)(3) receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (alpha4)(3)(beta2)(2) receptors. Furthermore, Zn2+ inhibition on (alpha4)(2)(beta2)(3) nAChRs is voltage-dependent, whereas it is not on (alpha4)(3)(beta2)(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 Zn(2+). Zn(2+) inhibition is mediated by a site located on the beta2(+)/alpha4(-) subunit interfaces on both receptor stoichiometries. alpha4(H195) and beta2(D218) are key determinants of this site. Zn2+ potentiation on (alpha4)(3)(beta2)(2) nAChRs is exerted by a site that resides on the alpha4(+)/alpha4(-) of this receptor stoichiometry. alpha4(H195) on the (-) side of the ACh-binding alpha4 subunit and alpha4(E224) on the (+) side of the non-ACh-binding alpha4 subunit critically contribute to this site. We also identified residues within the beta2 subunit that confer voltage dependency to Zn2+ inhibition on (alpha4)(2)(beta2)(3), but not on (alpha4)(3)(beta2)(2) nAChRs.
Moroni
Mirko
M
School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
Vijayan
Ranjit
R
Carbone
Anna
A
Zwart
Ruud
R
Biggin
Philip C
PC
Bermudez
Isabel
I
D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence / Q000502:physiology; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006801:Humans; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D009865:Oocytes; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011940:Receptor Aggregation / Q000187:drug effects / Q000502:physiology; D020239:Receptor Cross-Talk / Q000187:drug effects / Q000502:physiology; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology; D014982:Xenopus laevis; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology*
D008024:Ligands; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D015032:Zinc; D000109:Acetylcholine; D000409:Alanine