Expression and Subunit Interaction of Voltage-Dependent Ca2+Channels in PC12 Cells

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Abstract
Nerve growth factor (NGF)-induced differentiation in PC12 cells is accompanied by changes in the expression of voltage-dependent Ca2+channels. Ca2+channels are multimeric complexes composed of at least three subunits (α1, β, and α2δ) and are involved in neuronal migration, gene expression, and neurotransmitter release. Although attempts have been undertaken to elucidate NGF regulation of Ca2+channel expression, the changes in subunit composition of these channels during differentiation still remain uncertain. In the present study, patch-clamp recordings show that in addition to the previously documented L-type and N-type Ca2+currents, undifferentiated PC12 cells also express an ω-agatoxin-IVA-sensitive (P/Q-type) component. In addition, the corresponding mRNA encoding the pore-forming α1subunits for these channels (C, B, and A, respectively) was detected. Likewise, mRNA for three distinct auxiliary β subunits (1, 2, 3) were also found, β3protein being dominantly expressed. Immunoprecipitation experiments show that the N-type Ca2+channel is associated with either a β2or β3subunit and that NGF increases the channel expression without affecting its β subunit association. These results (1) indicate that the diversity of Ca2+currents in PC12 cells arise from the expression of three distinct α1and three different β subunit genes; (2) support a model for heterogenous β subunit association of the N-type Ca2+channel in a single cell type; and (3) suggest that the regulation of the N-type Ca2+channel during NGF-mediated differentiation involves an increase in the number of functional channels with no apparent changes in subunit composition.