We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of alpha and epsilon/epsilon' in all glioma cell lines analyzed; most, but not all cell lines expressed delta and zeta. No messages were found for the betaI and betaII isotypes of PKC in the tumor cells. Normal astrocytes expressed beta but not gamma. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKCbetaI and PKCbetaII isoforms inhibited BNaC2. Neither PKCepsilon nor PKCzeta or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKCbetaI and PKCbetaII mixture on BNaC2 activity was abolished by a 5-fold excess of a PKCepsilon and PKCzeta combination. PKC holoenzymes, PKCbetaI, PKCbetaII, PKCdelta, PKCepsilon, and PKCzeta phosphorylated BNaC2 in vitro. In patch clamp experiments, the combination of PKCbetaI and PKCbetaII inhibited the basally activated inward Na(+) conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2.