α7β2 Nicotinic Acetylcholine Receptors Assemble, Function, and Are Activated Primarily via Their α7-α7 Interfaces

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of alpha 7 and beta 2 subunits. We measured optical and electrophysiological properties of wildtype and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged alpha 7 and beta 2 subunits colocalize. Forster resonance energy transfer between fluorescently tagged subunits strongly suggested that alpha 7 and beta 2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function alpha 7 and beta 2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, alpha 7 beta 2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of alpha 7 nAChRs, although amplitudes of alpha 7 beta 2 nAChR-mediated, agonist-evoked currents were generally similar to 2-fold lower than those for alpha 7 nAChRs. It is noteworthy that alpha 7 beta 2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-beta-erythroidine that was not observed for alpha 7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the alpha 7-beta 2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower alpha 7 beta 2 nAChR current amplitudes and challenges in identifying the function of native alpha 7 beta 2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of beta 2 subunits within the alpha 7 beta 2 nAChRs.