Does enh facilitate protein kinase C modulation of N-type calcium current in sympathetic neurons?

Phorbol esters such as PMA activate protein kinase C (PKC), resulting in the block of N-type calcium (Ca) current inhibition via the membrane-delimited pathway. The site of convergence appears to occur at the I-II linker of the pore-forming subunit of the channel with phosphorylation antagonizing G-protein binding. The particular PKC isoform that modulates Ca current in superior cervical ganglion (SCG) neurons has not yet been identified. Maeno-Hikicki et al. (2003. Nature Neurosci. 6:468-475) demonstrated that the protein enigma homologue (enh) allows for specific anchoring of PKC[epsilon] to the carboxy-terminal tail of recombinant N-channels expressed in oocytes. The resulting channel complex augmented PKC modulation of Ca current, whereas inhibiting complex formation minimized modulation. Thus, PKC[epsilon] may be the primary PKC that mediates N-channel modulation. To determine whether this mechanism of N-channel modulation occurs in SCG neurons, we established which PKC isoforms are present in acutely dissociated neonatal SCG by Western blot analysis. At least 11 PKC isotorms are known and vary in their sensitivities to Ca and phorbol esters. The classical PKCs [alpha] [beta]I, [beta]II, and [gamma] are both Ca and phorbol ester sensitive, while the novel PKCs [delta], [epsilon], [theta], and [eta] are only phorbol ester sensitive and the atypical PKC's [zeta] and [iota]/[lambda] are insensitive to either Ca of phorbol esters. Our results are consistent with those found in cultured SCG neurons, confirming the presence of PKC [alpha], [beta]I, [beta]II, [delta], [epsilon], and [zeta] (Scholze et al. 2002. J. Neurosci. 22:5823-5832). We then established the presence of enh transcripts using RT-PCR. These preliminary findings indicate that both PKC[epsilon] and enh are present in the SCG. We are currently testing their role in PKC modulation of N-current in SCG neurons. (Supported by NIH grant RO1-NS34195.)