regulates the Kv4.2 potassium channel by direct phosphorylation of the pore-forming subunit. Am J Physiol Cell Physiol 290: C852-C861, 2006. First published October 26, 2005 doi:10.1152 doi:10. /ajpcell.00358.2005.2 is the primary pore-forming subunit encoding A-type currents in many neurons throughout the nervous system, and it also contributes to the transient outward currents of cardiac myocytes. A-type currents in the dendrites of hippocampal CA1 pyramidal neurons are regulated by activation of ERK/MAPK, and Kv4.2 is the likely pore-forming subunit of that current. We showed previously that Kv4.2 is directly phosphorylated at three sites by ERK/MAPK (T602, T607, and S616). In this study we determined whether direct phosphorylation of Kv4.2 by ERK/ MAPK is responsible for the regulation of the A-type current observed in neurons. We made site-directed mutants, changing the phosphosite serine (S) or threonine (T) to aspartate (D) to mimic phosphorylation. We found that the T607D mutation mimicked the electrophysiological changes elicited by ERK/MAPK activation in neurons: a rightward shift of the activation curve and an overall reduction in current compared with wild type (WT). Surprisingly, the S616D mutation caused the opposite effect, a leftward shift in the activation voltage. K ϩ channel-interacting protein (KChIP)3 ancillary subunit coexpression with Kv4.2 was necessary for the T607D effect, as the T607D mutant when expressed in the absence of KChIP3 was not different from WT Kv4.2. These data suggest that direct phosphorylation of Kv4.2 at T607 is involved in the dynamic regulation of the channel function by ERK/MAPK and an interaction of the primary subunit with KChIP is also necessary for this effect. Overall these studies provide new insights into the structure-function relationships for MAPK regulation of membrane ion channels. K ϩ channel-interacting protein; kinase; neurons; A-type current MANY STUDIES HAVE SHOWN THAT ERK is important for regulation of neuronal function, particularly playing a role in the regulation of synaptic plasticity and long-term memory formation (3,13,14,30,46,48). Considerable evidence is accumulating that ERK activation plays a role in the regulation of postsynaptic excitability, specifically operating in the context of synaptic plasticity (40,46,48). One potential mechanism of this regulation by ERK is indirect, by long-term modulation of cell properties through the control of gene transcription and regulation of channel gene expression (9). Another possible mechanism by which ERK might modulate neuronal excitability is through direct regulation of membrane ion channels that regulate the membrane potential and thereby intrinsic membrane properties.In our recent studies, we have focused on regulation of ion channels by ERK because modulation of excitability may be a critical factor that ultimately controls the induction of longlasting changes in synaptic strength. One possible direct target of ERK is the K ϩ channel Kv4.2, which encodes a transient A-type K ϩ current that...

Small conductance, Ca2؉ -activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 M) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxylterminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser 465 (minor site) and three amino acid residues Ser 568 , Ser 569 , and Ser 570(major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.The small conductance, Ca 2ϩ -activated K ϩ (SK) 2 channels are found in both neuronal and non-neuronal tissue (1). Functionally, the SK channels are best characterized in the central nervous system. Three genes encode the SK channel subunits (SK1, SK2, and SK3) in mammalian brain (2). SK channels are blocked by the bee venom toxin, apamin, although SK1 is slightly less sensitive than SK2 and SK3 (2-4). In neurons throughout the nervous system, the apamin-sensitive SK channels modulate firing frequency by contribution to the afterhyperpolarization (AHP) that follows a single or a train of action potentials (5-7). SK2 is thought to specifically underlie the medium AHP current (I mAHP ) in hippocampal CA1 pyramidal cells (8). The I mAHP is Ca 2ϩ -dependent with a time constant of 100 -250 ms and sensitivity to apamin (9 -11).The apamin-sensitive I mAHP modulates instantaneous firing rates and sets the interspike duration in action potential trains to produce spike frequency adaptation (12). In addition, SK channels are localized to the dendrites of pyramidal cells in hippocampal area CA1 and pyramidal neurons of the lateral amygdala where they function to shape synaptic potentials and limit Ca 2ϩ influx through NMDA receptors (13, 14) as well as plateau potentials evoked by exogenous glutamate application (15). Other studies have linked overexpression of SK2 and enhancement of the I mAHP in hippocampus with neuroprotection (16), attenuation of hippocampal LTP, and memory deficits (16, 17). These functions have major implications for a rol...

Extracelluar signal‐regulated kinase (ERK) pathway activation has been demonstrated following convulsant stimulation; however, little is known about the molecular targets of ERK in seizure models. Recently, it has been shown that ERK phosphorylates Kv4.2 channels leading to down‐regulation of channel function, and substantially alters dendritic excitability. In the kainate model of status epilepticus (SE), we investigated whether ERK phosphorylates Kv4.2 and whether the changes in Kv4.2 were evident at a synaptosomal level during SE. Western blotting was performed on rat hippocampal whole cell, membrane, synaptosomal, and surface biotinylated extracts following systemic kainate using an antibody generated against the Kv4.2 ERK sites and for Kv4.2, ERK, and phospho‐ERK. ERK activation was associated with an increase in Kv4.2 phosphorylation during behavioral SE. During SE, ERK activation and Kv4.2 phosphorylation were evident at the whole cell and synaptosomal levels. In addition, while whole‐cell preparations revealed no alterations in total Kv4.2 levels, a decrease in synaptosomal and surface expression of Kv4.2 was evident after prolonged SE. These results demonstrate ERK pathway coupling to Kv4.2 phosphorylation. The finding of decreased Kv4.2 levels in hippocampal synaptosomes and surface membranes suggest additional mechanisms for decreasing the dendritic A‐current, which could lead to altered intrinsic membrane excitability during SE.

We evaluated a role for the nuclear factor-kappa B (NF-jB) pathway in the regulation of seizure susceptibility and transcriptional activation during prolonged, continuous seizures (status epilepticus). Using two functionally distinct NF-jB inhibitors we observed a decrease in latency to onset of kainate-induced seizures and status epilepticus. To assess NFjB transcriptional activation, we evaluated inhibitor kappa B alpha (IjBa) and brain-derived neurotrophic factor (bdnf) gene targets. Inhibition of the NF-jB signaling pathway significantly attenuated the increases in IjBa and bdnf mRNA levels that occurred during prolonged seizure activity, suggesting that the NF-jB pathway was involved in the up-regulation of these transcripts during status epilepticus. DNA-binding studies and chromatin immunoprecipitation assays using hippocampal extracts from animals with status epilepticus revealed that NFjB subunits were associated with the candidate jB-binding elements within promoter 1 of the bdnf gene. The pattern of association was different for the p50 and p65 subunits supporting complex NF-jB modifications within promoter 1. In summary, our findings provide additional insights into the role of NF-jB transcriptional regulation in hippocampus following status epilepticus and suggest that NF-jB pathway activation contributes to seizure susceptibility.

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 protein kinase C (PKC) activation, and the K+ channel subunit, Kv4.2, is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In this study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular amino (N)- and carboxyl (C)-termini of Kv4.2 glutathione S-transferase (GST) fusion protein revealed that the Kv4.2 C-terminal was phosphorylated by PKC, while the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Serine (Ser) 447 and Ser537. A phospho-site specific antibody showed that phosphorylation at the Ser537 site increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that alanine mutation to block phosphorylation at both of the PKC sites increased surface expression compared to wildtype Kv4.2. Electrophysiological recordings of the wildtype and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 revealed no significant difference in the half activation or inactivation voltage of the channel. Interestingly, the Ser537 site lies within a possible extracellular regulated kinase (ERK)/mitogen activated protein kinase (MAPK) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC 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.