Nicotinic acetylcholine receptor (nAChR) ␣4 and ␤2 subunits assemble in two alternate stoichiometries to produce (␣4␤2) 2 ␣4 and (␣4␤2) 2 ␤2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at ␣4(؉)/␤2(؊) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either ␣4 or ␤2 as auxiliary subunits produces isoform-specific ␣4(؉)/␣4(؊) or ␤2(؉)/␤2(؊) interfaces. Using fully concatenated (␣4␤2) 2 ␣4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the ␣4(؉)/ ␣4(؊) interface that accounts for isoform-specific agonist sensitivity of the (␣4␤2) 2 ␣4 nAChR. The additional site resides in a region that also contains a potentiating Zn 2؉ site but is engaged by agonists to contribute to receptor activation. By engineering ␣4 subunits to provide a free cysteine in loop C at the ␣4(؉)␣4(؊) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the ␣4(؉)/(␣4(؊) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the ␣4(؉)/ ␣4(؊) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (␣4␤2) 2 ␣4 nAChRs.

Edited by F. Anne StephensonAllosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using ␣4␤2 nicotinic acetylcholine receptors and the ␣4␤2-selective positive modulators 17␤-estradiol (␤EST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiating ␤EST is in the C-terminal (post-M4) region of the ␣4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) ␣-helices of the ␣4 subunit. We found that the binding sites for ␤EST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and Phe 170 of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Tyr 309 ) and Phe 167 , a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating ␤EST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.The ␣4␤2 nicotinic acetylcholine receptor (nAChR) 2 belongs to the superfamily of pentameric ligand-gated ion channels (pLGICs). In humans, this superfamily comprises the Cys loop receptors (including muscle and neuronal nAChRs, 5-HT 3 , GABA A , and glycine receptors), which mediate all fast central nervous system synaptic inhibition and much of fast peripheral excitation. Cys loop receptor subunits assemble as a pentamer of identical (homomeric channels) or different (heteromeric channels) subunits around a central ion channel. The individual subunits have a large extracellular N-terminal domain (ECD) that consists of 10 ␤-strands (␤1-␤10) folded into a ␤-sandwich and a transmembrane domain (TMD) with four transmembrane ␣ helices (M1-M4) connected by linkers (M1-M2, M2-M3, and M3-M4), as well as intracellular domains and a highly variable extracellular C-terminal (post-M4) region (1). There are 2-5 neurotransmitter binding sites at subunit interfaces within the ECD, and these sites are functionally coupled to the transmembrane ion channel located ϳ50 Å away. In the nAChR, the subunit contributing the principal face of the agonist binding site (an ␣ subunit) couples agonist-triggered agonist binding site movements to channel gating (2, 3). The coupling is achieved at the ECD-TMD interface by a principal pathway that couples the pre-M1 region in the ECD to the M2-M3 linker through the ␤1-␤2 loop (4) and the canonical FPF motif of the ␤6-...

BACKGROUND AND PURPOSENicotinic ACh (α4β2)2α4 receptors are highly prone to desensitization by prolonged exposure to low concentrations of agonist. Here, we report on the sensitivity of the three agonist sites of the (α4β2)2α4 to desensitization induced by prolonged exposure to ACh. We present electrophysiological data that show that the agonist sites of the (α4β2)2α4 receptor have different sensitivity to desensitization and that full receptor occupation decreases sensitivity to desensitization. EXPERIMENTAL APPROACHTwo-electrode voltage-clamp electrophysiology was used to study the desensitization of concatenated (α4β2)2α4 receptors expressed heterologously in Xenopus oocytes. Desensitization was assessed by measuring the degree of functional inhibition caused by prolonged exposure to ACh, as measured under equilibrium conditions. We used the single-point mutation α4W182A to measure the contribution of individual agonist sites to desensitization. KEY RESULTS(α4β2)2α4 receptors are less sensitive to activation and desensitization by ACh than (α4β2)2β2 receptors. Incorporation of α4W182A into any of the agonist sites of concatenated (α4β2)2α4 receptors decreased sensitivity to activation and desensitization but the effects were more pronounced when the mutation was introduced into the α4(+)/α4(−) interface. CONCLUSIONS AND IMPLICATIONSThe findings suggest that the agonist sites in (α4β2)2α4 receptors are not functionally equivalent. The agonist site at the α4(+)/α4(−) interface defines the sensitivity of (α4β2)2α4 receptors to agonist-induced activation and desensitization. Functional differences between (α4β2)2α4 and (α4β2)2β2 receptors might shape the physiological and behavioural responses to nicotinic ligands when the receptors are exposed to nicotinic ligands for prolonged periods of times.

Background:The ␣4/␤2 and ␣4/␣4 interfaces of the (␣4␤2) 2 ␣4 nicotinic acetylcholine receptor house structurally different agonist sites. Results: Agonists of a certain size cannot bind the ␣4/␣4 interface, which decreases efficacy. Conclusion:The ability to bind all agonist sites in (␣4␤2) 2 ␣4 receptors critically influences agonist efficacy. Significance: The finding adds a new level of complexity to structural mechanisms governing agonist efficacy.

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