This work aims at the characterization of the modulation of gene expression and activity of nicotinic acetylcholine receptors during neuronal maturation of PC12 cells. The rat pheochromocytoma cell line (PC12) is a widely accepted model system for studying neuronal differentiation in vitro. In the presence of basic fibroblast growth factor (bFGF) and dibutyril-cAMP, PC12 cells differentiate to neurons and express functional nicotinic acetylcholine receptors (nAChRs), as determined by voltage-clamp whole-cell recording in the presence of the stable acetylcholine analog carbamoylcholine (CCh). Increasing whole-cell currents in PC12 cells to CCh application were observed during differentiation, ranging from 1400 pA on the first day to 7200 pA on the seventh day. RT-PCR and real-time PCR were performed to determine whether the change in receptor activity is due to modulation of gene expression. In previous studies we have determined that differentiating PC12 cells express mRNAs coding for alpha 3, 5, 7 and beta 2, 4 nAChR subunits.
Modulation of alpha-7 subunit gene expression and activity was studied during the course of differentiation.  On the first day, we have detected a maximum of alpha 7 subunit gene expression. However, maximal whole-cell current responses to CCh application with rapid desensitization rates were recorded on day 2, indicating the presence of homomeric receptors formed by alpha 7 subunits. The participation of alpha 7 receptors in the whole-cell current response induced by CCh, was confirmed in the presence of their specific inhibitor methyllycaconitine MLA (10 nM). MLA co-application with CCh compared to control experiments in the presence of CCh alone resulted in an inhibition factor of two on day two, compared to almost no inhibition on days one and seven by MLA.
These data indicate that alpha-7 receptor expression and activity are needed at the beginning of neuronal differentiation, and are not any more present when already functional neurons have been formed.