Based on the characteristics of differentiated NG108-15 cells (cell membrane excitability, acetylcholine release, and activities of choline acetyltransferase and acetylcholinesterase), NG108-15 cells are extensively used to explore neuronal functions as a cholinergic cell line. In the present study, differentiation-induced alterations of voltage-gated Ca2+ channel mRNA, protein, and current were investigated in the NG108-15 cells. Real-time PCR, Western blot, and whole-cell patch-clamp data showed that differentiation caused mRNA, protein, and ion current changes of all Ca2+ channel subunits. However, the changes of mRNA, protein, and ion current are inconsistent in all Ca2+ channel subunits. Especially, P/Q- and R-type Ca2+ channel proteins do not form the functional P/Q- and R-type Ca2+ channels even if the mRNA and protein of P/Q- and R-type Ca2+ channels can be detected in NG108-15 cells. These results indicate that differentiation can modulate gene transcription, protein translation, and post-translation of the Ca2+ channels to induce the alteration of the Ca2+ ion currents in NG108-15 cells. From these data, we understand that combining real-time PCR, Western blot, and patch-clamp techniques can comprehensively unveil the modulation of the Ca2+ channels. (C) 2012 Elsevier Inc. All rights reserved.