Kv4.2 is a locus for PKC and ERK/MAPK cross-talk

Transient outward K(+) currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K(+)-channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Ill channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that Ill directly phosphorylates the Kv4.2 channel protein. hi vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by Ill whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser(447) and Ser(537). A phospho-site-specific antibody showed that phosphorylation at the Ser(537) site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser(447) and Ser(537) in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser(537) lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by Ill enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.