Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2* Nicotinic Acetylcholine Receptors

Abstract

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric ␣4␤2* nAChRs typically have two ACh binding sites at ␣4/␤2 interfaces and a fifth accessory subunit surrounding the central cation channel. ␤2 accessory subunits do not form ACh binding sites, but ␣4 accessory subunits do at the ␣4/␣4 interface in (␣4␤2) 2 ␣4 nAChRs. ␣5 and ␤3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that ␣5/␣4 and ␤3/␣4 interfaces formed ACh binding sites in (␣4␤2) 2 ␣5 and (␣4␤2) 2 ␤3 nAChRs. The ␣4/␣5 interface in (␤2␣4) 2 ␣5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30 -50%. However, site-selective agonists NS9283 (for the ␣4/␣4 site) and sazetidine-A (for the ␣4/␤2 site) did not act on the ACh sites formed by the ␣5/␣4 or ␤3/␣4 interfaces. This suggests that unorthodox sites formed by ␣5 and ␤3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders. Nicotinic acetylcholine receptors (nAChRs)2 are ACh-gated ion channels formed from five homologous subunits organized like barrel staves around a central cation channel (1). There are homomeric nAChRs and heteromeric nAChRs. Homomeric ␣7 nAChRs have five ␣7 subunits with ACh binding sites between each of the five ␣7/␣7 interfaces. All nAChR subunits exhibit basic homology throughout their sequences, indicating that all nAChRs evolved from a common ancestor (1, 2). Heteromeric nAChR agonist binding sites typically form at the interface between the primary (ϩ) side of an ␣ subunit, characterized by the presence of a C loop that closes over the site when the nAChR is activated by an agonist, and the complementary (Ϫ) side of a ␤2 or ␤4 subunit (3). Two such ACh binding sites assemble with an accessory subunit in a stoichiometry such as (␣4␤2) 2 ␤2 (4, 5).The (␣4␤2) 2 ␣4 stoichiometry contains an unorthodox ACh binding site at the ␣4/␣4 interface (6, 7). When this low affinity unorthodox site is bound by ACh or NS9283, an agonist specific for this site, nAChRs activate 3-4-fold more efficiently (8, 9). Blocking the ␣4/␣4 site through alkylation of a cysteine introduced in the minus face of the ␣4 subunit blocked the activity of ACh and NS9283 (9). Histidine 142 (this is position 116 in the mature ␣4 peptide sequence) on the minus side of ␣4 is critical for the binding of NS9283 (10). Sazetidine-A is a high affinity agonist selective for ␣4/␤2 sites that cannot bind to the ␣4/␣4 interface because histidine 142 on the...

Nicotinic acetylcholine receptors in neuropathic and inflammatory pain

“…These studies concluded that (α4) 2 (β2) 3 nAChRs showed high sensitivity (HS) to agonists ligands, whereas those with an (α4) 3 (β2) 2 stoichiometry showed low sensitivity (LS). It has also been shown that a 3rd noncanonical ligand‐binding site is present between the α4–α4 interface of the (α4) 3 (β2) 2 LS stoichiometry, the α5–α4 interface of the (α4) 2 (β2) 2 α5 subtype, and the β3–α4 interface in the (α4) 2 (β2) 2 β3 subtype . Interestingly, some agonist ligands are excluded from these noncanonical sites.…”

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors

“…Recently, a third ACh site was identified at the α4/α4 subunit interface in (α4β2) 2 α4 nAChRs (Harpsoe et al , ; Mazzaferro et al , ). Subsequently, additional ACh sites were found at α5/α4, β3/α4 and α4/α5 subunit interfaces (Jin et al , ; Wang et al , ; Jain et al , ). These sites are referred to as unorthodox sites.…”

“…Subunit interfaces that form ACh‐binding sites are noted. α5 and β3 subunits form an ACh‐binding site with α4 subunit (Jain et al , ), and it is to be determined whether they do so with other α subunits. (B) Illustration of different stoichiometric forms of (α4) 2 (β2) 2 α5 expressed from subunit concatamers (Jain et al , ).…”

“…α5 and β3 subunits form an ACh‐binding site with α4 subunit (Jain et al , ), and it is to be determined whether they do so with other α subunits. (B) Illustration of different stoichiometric forms of (α4) 2 (β2) 2 α5 expressed from subunit concatamers (Jain et al , ). In the ACh‐binding site formed at the interface with α4, α5 can act either as an α subunit when expressing β2‐(AGS) 6 ‐α4 with free α5 subunit (left) or as a β subunit when expressing α4‐(AGS) 6 ‐β2 with free α5 (right) (Jain et al , ).…”

“…Linked subunits define both subunit composition and order (Zhou et al , ; Kuryatov and Lindstrom, ; Kuryatov et al , ; Wang et al , ) (Figure B). This method has been used to understand the pharmacology of complex nAChRs (Kuryatov and Lindstrom, ; Jin et al , ; Mazzaferro et al , , ) as well as to identify agonist‐binding sites at subunit interfaces (Harpsoe et al , ; Mazzaferro et al , , ; Wang et al , ; Jain et al , ). Although the two β/α subunit interfaces cannot form ACh sites, the accessory subunit can form an ACh site when it is α2, α3, α4, α5, α6 or β3 (Figure A).…”