Stoichiometry and kinetics of activation and potentiation of nicotinic alpha7beta2 heteromeric receptors

Abstract

The α7 nicotinic receptor (nAChR) is a promising drug target for neurological and inflammatory disorders. It has been considered the homomeric member of the family. The recent discovery of α7β2 receptor in brain led to the urgent need of its functional characterization. Our main goal is to determine the stoichiometry of the heteromeric α7β2 receptor and its activation and potentiation profile. We generated receptors with fixed stoichiometry by two different approaches. One involved the generation of concatemeric α7β2 pentamers of different stoichiometries, and the other involved co-expression of unlinked α7 and β2 subunits, with the α7 subunit carrying a reporter mutation. Receptors were expressed in mammalian cells and function was evaluated by single-channel recordings. We found that α7 can assemble with one, two or three β2 subunits to form functional receptors. As the number of β2 subunits in the pentamer increases, the durations of openings and activation episodes, called bursts, increase progressively whereas channel conductance remains constant. We proposed that the prolonged bursts observed for α7β2 can be used as the signature of the presence of heteromeric receptors in native tissues. The prolonged activation episodes and reduced desensitization of α7β2 may have an important impact on calcium-dependent intracellular signaling and neuronal excitability. By using mutant subunits, we demonstrated that activation of α7β2 occurs through the α7/α7 binding-site interface. Among α7 positive allosteric modulators (PAMs), which emerge as novel therapeutic tools, type I PAMs were more selective for α7 than for α7β2 whereas PNU-120596, a type II PAM, similarly potentiated all α7-containing receptors. Statistically significance differences were established at p-values<0.05.This first single-channel study of α7β2 provides basis for deciphering the role and functional location of this novel receptor and opens doors for the development of selective therapeutic drugs