In this study we showed that the C-terminal of Kv4.2 is phosphorylated by PKC at two sites, Ser447 and Ser537. Using a phospho-selective antibody against the Ser537 PKC site within Kv4.2 we determined an increase in Kv4.2 phosphorylation at this site following PKC pathway activation in hippocampal slices, indicating that PKC couples to Kv4.2 phosphorylation in a system where it is natively expressed. Additional functional data were evident in the COS7 expression system using phospho-site mutants. These studies suggested that PKC phosphorylation of Kv4.2 regulates surface expression of the channel. These studies showed that when the PKC sites within Kv4.2 were mutated to alanines to block phosphorylation there was a significant increase in surface expression of Kv4.2 compared to wildtype channels. In addition, PKC phosphorylation of Kv4.2 enhanced ERK phosphorylation of the channel in vitro, suggesting the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.
Mimicking or blocking phosphorylation of Kv4.2 at the PKC sites (Ser447 and Ser537) did not modulate the kinetics of the Kv4.2-mediated current, as demonstrated by electrophysiological recordings of the DD and AA Kv4.2 channel mutants. Specifically, there were no differences in the half-activation and -inactivation voltages between the WT and double mutant channels (AA and DD). However, as noted above we demonstrated that surface expression of the AA mutant increased as indicated by an enhanced peak current in oocytes expressing the AA mutant channel. This suggests that basal phosphorylation of the channel by PKC (or ERK/MAPK) reduces trafficking or maintenance of the channel in the membrane.
We found that prior phosphorylation of the Kv4.2 C-terminal by PKC caused an increase in ERK phosphorylation at two of the three ERK/MAPK sites, Thr602, Thr607, but not Ser616 [26
]. This is interesting in the context of our previously reported results which showed that phosphorylation at 602 and 607 sites had an overall inhibitory effect on the current, which mimicked the effect of ERK/MAPK activation seen in the dendrites of CA1 pyramidal neurons [11
], whereas phosphorylation at Ser616 caused an opposite effect [24
]. The Thr607Asp (T607D) mutation mimicked the effect of ERK seen in neurons, which includes a right shift in the activation curve and an overall decrease in current. This suggests that at least the Thr607 site is the relevant site phosphorylated in response to PKC activation in neurons [11
]. Moreover, the Thr607 site effect is dominant when all 3 sites are phosphorylated. Phosphorylation of the C-terminal by PKC prior to ERK may augment phosphorylation at the Thr607 site, increasing the balance of phosphorylation at that site over the Ser616 site to ensure a decrease in current. This does not preclude phosphorylation at Ser616 in response to different physiological stimulation.
Previous data suggest that filamin is a scaffold protein that links Kv4.2 and the actin cytoskeleton. Furthermore, mutation of amino acids 601-604 region in the Kv4.2 C-terminal to alanines completely abolished the interaction of Kv4.2 and filamin in a yeast two-hybrid assay [37
]. This suggests that this region is important for the interaction of Kv4.2 with filamin and possibly for Kv4.2 localization and stabilization in the membrane. Phosphorylation of the ERK amino acid residues in this region on the Kv4.2 C-terminal (Thr602 and Thr607) may alter the interaction of filamin and Kv4.2 such that Kv4.2 becomes unstable at the surface membrane. Enhanced phosphorylation of these sites mediated by phosphorylation by PKC may play a role in ‘tagging’ Kv4.2 subunits for internalization. Further studies are necessary to determine the exact role of the interaction of PKC and ERK phosphorylation sites and the interaction with filamin.
Kv4.2 is the primary subunit that contributes to IA
in the dendrites of CA1 pyramidal cells, and the threshold for induction of synaptic plasticity in the CA1 pyramidal cell dendrites is lower in the Kv4.2 KO animals as compared to control [5
]. This suggests that a decrease in IA
can augment induction of synaptic plasticity. IA
is regulated by activation of PKC, and the effect is blocked by the ERK/MAPK inhibitor U0126 [11
]. This suggests the possibility that direct phosphorylation of the Kv4.2 subunit by both PKC and ERK/MAPK may modulate the current. Indeed, our phospho-site specific antibody demonstrates that Kv4.2 is phosphorylated at Ser537 in the hippocampus in response to PKC activation.
We have previously shown that the Kv4.2 current is modulated by direct phosphorylation of Kv4.2 by ERK/MAPK [24
]. These data provide further support for interplay in the regulation of the A-type currents by various kinases and provide another possibility of Kv4.2 as a site of signal integration in neurons and cardiac myocytes. The modulation of ERK phosphorylation of the channel by PKC phosphorylation may be a mechanism of fine-tuning the current in response to a physiological stimulus. Moreover, a greater decrease in current mediated by both direct phosphorylation of the channel by PKC and ERK/MAPK may be a mechanism to dynamically regulate current amplitude and induction of synaptic plasticity.