CHRFAM7A alters binding to the neuronal alpha-7 nicotinic acetylcholine receptor

Introduction: CHRFAM7A is a uniquely-human gene that encodes a human-specific variant of the alpha -7 nicotinic acetylcholine receptor (alpha 7nAchR). While the homopentameric alpha 7nAChR consists of 5 equal subunits, previous studies demonstrated that CHRFAM7A expression disrupts the formation of alpha 7nAChR homopentamers. Here we use a rat neuronal cell line expressing CHRFAM7A and a transgenic mouse expressing CHRFAM7A to define the alpha-bungarotoxin (alpha-BTX) binding in vitro and in vivo. Methods: Rat PC12 cells were stably transfected with human CHRFAM7A. alpha-BTX, a protein that irreversibly binds the alpha 7nAchR, was utilized to assess the capacity for CHRFAM7A to interfere with alpha 7AchR subunits using immunohistochemistry and flow cytometry. To evaluate the effects of CHRFAM7A on alpha 7nAchR at the neuromuscular junction in vivo, transgenic mice were engineered to express the uniquely human gene CHRFAM7A under the control of the EF1-alpha promoter. Using this model, muscle was harvested and CHRFAM7A and CHRNA7 gene expression evaluated by PCR. Binding of alpha-BTX to the alpha 7nAchR in muscle was compared in sibling-matched wild-type C57 mice by immunostaining the neuromuscular junction using alpha-BTX and neurofilament antibodies. Results: Expression of CHRFAM7A in transfected, but not vector cells, was confirmed by PCR and by immunoblotting using an antibody we raised to a peptide sequence unique to CHRFAM7A. CHRFAM7A decreased alpha-BTX binding as detected by immunohistochemistry and flow cytometry. In vivo, alpha-BTX co-stained with neurofilament at the neuromuscular junction in wild-type mice, however, alpha-BTX staining was decreased at the neuromuscular junction of CHRFAM7A transgenic mice. Conclusion: CHRFAM7A expression interferes with the binding of alpha 7nAchR to alpha-BTX. Understanding the contribution of this uniquely human gene to human disease will be important in the identification of potential therapeutic targets.