The modulation of Ca2+ channel activity by protein kinases contributes to the dynamic regulation of neuronal physiology. Using the transient expression of a family of neuronal Ca2+ channels, we have identified several factors that contribute to the PKC-dependent modulation of Ca2+ channels. First, the nature of the Ca2+ channel alpha 1 subunit protein is critical. Both alpha 1B and alpha 1E channels exhibit a 30%-40% increase in peak currents after exposure to phorbol esters, whereas neither alpha 1A nor alpha 1C channels are significantly affected. This up-regulation can be mimicked for alpha 1E channels by stimulation of a coexpressed metabotropic glutamate receptor (type 1 alpha) through a PKC-dependent pathway. Second, PKC-stimulated up-regulation is dependent upon coexpression with a Ca2+ channel beta subunit. Third, substitution of the cytoplasmic domain I-II linker from alpha 1B confers PKC sensitivity to alpha 1A channels. The results provide direct evidence for the modulation of a subset of neuronal Ca2+ channels by PKC and implicate alpha 1 and beta subunit interactions in regulating channel activity via second messenger pathways.