Contributions of Torpedo Nicotinic Acetylcholine Receptor γTrp-55 and δTrp-57 to Agonist and Competitive Antagonist Function

Abstract

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the ␣-␥ and ␣-␦ subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[3 H]tubocurarine (dTC) identified two tryptophans, ␥Trp-55 and ␦Trp-57, as the primary sites of photolabeling in the non-␣ subunits. To characterize the importance of ␥Trp-55 and ␦Trp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the ␦W57L and ␥W55L mutations decreased acetylcholine (ACh) binding affinity by 20-and 7,000-fold respectively. For the wild-type, ␥W55L, and ␦W57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the ␥W55L mutant, dTC binding at the ␣-␥ site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with ␥ Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening. The nicotinic acetylcholine receptor (nAChR)1 from Torpedo electric organ and vertebrate skeletal muscle is a pentameric transmembrane protein composed of four homologous subunits with a stoichiometry of ␣ 2 ␤␥␦ (reviewed in Refs. 1-3). The nAChR contains two binding sites for agonists and competitive antagonists, located at the ␣-␥ and ␣-␦ subunit interfaces (4, 5). The two sites are nonequivalent, and many competitive antagonists bind with high affinity to only one of the sites (6, 7). Affinity labeling and mutational analyses provide evidence that amino acids from three discrete regions of ␣ subunit primary structure and from three (or more) regions of the ␥ (or ␦) subunit contribute to the structure of the binding sites (reviewed in Refs. 8 and 9).Several experimental approaches have identified residues in both ␥ and ␦ subunits that contribute to the binding sites for agonists or competitive antagonists. Photoaffinity labeling using d-[3 H]tubocurarine (dTC), a competitive antagonist, and [ 3 H]nicotine, an agonist, established that ␥Trp-55 is located near the agonist binding site in Torpedo nAChR (10, 11), and [ 3 H]dTC also reacted with ␦Trp-57, the corresponding position in the ␦ subunit, as well as with ␥Tyr-111/␥Tyr-117 (12). Substitution of ␥Trp-55 by leucine (␥W55L) caused a 10-fold decrease in dTC potency as an inhibitor of ACh-induced currents for Torpedo nAChRs expressed in Xenopus oocytes (13). A heterobifunctional cross-linker ϳ9 Å in length cross-linked ␣Cys-192/␣Cys-193 in Torpedo nAChR to a residue in the ␦ subunit identified as ␦Asp-180 (14, 15), and the mutation ␦D180N in the ␦ subunit of mouse muscle nAC...

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

“…In Torpedo AChRs, a leucine substitution at either ⑀Trp-55 or ␦Trp-57 increases the response to EC 50 and increases the equilibrium dissociation constants for both agonists and antagonists (10). Replacement of Trp-54 in ␣7 neuronal AChRs (homologous to ␥Trp-55/␦Trp-57 in muscle AChRs) by Phe, Ala, or His produces similar effects (15), as do substitutions at homologous positions in other Cys loop receptors (16 -18).…”

“…In Torpedo, ␥Trp-55 and ␦Trp-57 are sites of photo-incorporation of the competitive antagonist d-tubocurarine (9,10), and ␥Trp-55 is labeled by the agonist nicotine (11). Also, constitutively active AChRs are formed following the incorporation of a series of tethered quaternary ammonium derivatives at ␣Trp-149, ␣Tyr-93, and ␥Trp-55/␦Trp-57 (12,13).…”

The nicotinic receptor ligand binding domain

“…Measurements of agonist affinity following mutagenesis, as expected, revealed large decreases in affinity following mutation of the aromatic residues, ␣W149, ␣Y190, ␣Y93, and ␣Y198 (Galzi et al, 1991b;O'Leary and White, 1992;Sine et al, 1994). However, even the question of which subunit is involved was uncertain because large decreases in agonist affinity were also observed following mutation of ␦W57, ␦S36, ␦I178, and ␦D175 (Czajkowski, et al 1993;Prince and Sine, 1996;Xie and Cohen, 2001). Perhaps the strongest evidence for the identity of the primary ACh docking site came from studies of unnatural mutations in which varying numbers of fluorines were substituted on tryptophan at position 149 in the ␣-subunit, and EC 50 s for receptor activation were compared to quantum mechanical calculations of interaction energies between the tryptophan analogs and a cation (Zhong et al, 1998).…”

Nicotinic Acetylcholine Receptors

“…Their order around the pore is most likely to be α, γ, α, β, δ going in the clockwise sense and viewed from the direction of the synaptic cleft. Opening of the channel occurs upon binding of ACh to both α subunits (α γ and α δ ) at sites that are at, or close to, the interfaces made with neighbouring γ and δ subunits ( Karlin, 1993;Sine et al, 1995a;Xie & Cohen, 2001). These sites are shaped by three separate regions of the polypeptide chain (Corringer et al, 2000), and include the so-called C-loop (see below).…”

“…The most important residues in neighbouring subunits that influence ACh binding are W57 of the δ subunit and the homologous W55 of the γ subunit (Chiara & Cohen, 1997;Xie & Cohen, 2001). The 'minus side' of the AChBP interface, shown in Figure 6B, would be the γ/ε or the δ subunit in the receptor, but in the AChBP it is another identical subunit.…”