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.

Neuroimmune dysregulation is implicated in neuropsychiatric disorders including schizophrenia. As the blood−brain barrier is the immunological interface between the brain and the periphery, we investigated whether this vascular phenotype is intrinsically compromised in the most common genetic risk factor for schizophrenia, the 22q11.2 deletion syndrome (22qDS). Blood−brain barrier like endothelium differentiated from human 22qDS+schizophrenia-induced pluripotent stem cells exhibited impaired barrier integrity, a phenotype substantiated in a mouse model of 22qDS. The proinflammatory intercellular adhesion molecule-1 was upregulated in 22qDS+schizophrenia-induced blood–brain barrier and in 22qDS mice, indicating compromise of the blood–brain barrier immune privilege. This immune imbalance resulted in increased migration/activation of leucocytes crossing the 22qDS+schizophrenia blood−brain barrier. We also found heightened astrocyte activation in murine 22qDS, suggesting that the blood−brain barrier promotes astrocyte-mediated neuroinflammation. Finally, we substantiated these findings in post-mortem 22qDS brain tissue. Overall, the barrier-promoting and immune privilege properties of the 22qDS blood–brain barrier are compromised, and this might increase the risk for neuropsychiatric disease.

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.