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.

SUMMARYPurpose: Kv4.2 subunits contribute to the poreforming region of channels that express a transient, A-type K + current (A-current) in hippocampal CA1 pyramidal cell dendrites. Here, the A-current plays an important role in signal processing and synaptic integration. Kv4.2 knockout mice show a near elimination of the A-current in area CA1 dendrites, producing increased excitability in this region. In these studies, we evaluated young adult Kv4.2 knockout mice for spontaneous seizures and the response to convulsant stimulation in the whole animal in vivo and in hippocampal slices in vitro. Methods: Electroencephalogram electrode-implanted Kv4.2 knockout and wild-type mice were observed for spontaneous behavioral and electrographic seizures. The latency to seizure and status epilepticus onset in Kv4.2 knockout and wild-type mice was assessed following intraperitoneal injection of kainate. Extracellular field potential recordings were performed in hippocampal slices from Kv4.2 knockout and wild-type mice following the bath application of bicuculline. Results: No spontaneous behavioral or electrographic seizures were observed in Kv4.2 knockout mice. Following kainate, Kv4.2 knockout mice demonstrated a decreased seizure and status epilepticus latency as well as increased mortality compared to wild-type littermates. The background strain modified the seizure susceptibility phenotype in Kv4.2 knockout mice. In response to bicuculline, slices from Kv4.2 knockout mice exhibited an increase in epileptiform bursting in area CA1 as compared to wild-type littermates. Discussion: These studies show that loss of Kv4.2 channels is associated with enhanced susceptibility to convulsant stimulation, supporting the concept that Kv4.2 deficiency may contribute to aberrant network excitability and regulate seizure threshold. KEY WORDS: A-type K channel, Channelopathies, Seizures, Status epilepticus, Mouse.Kv4.2 subunits compose the pore-forming channel that contributes to the transient, rapidly activating and inactivating outward K + current (A-current) in CA1 pyramidal cell dendrites (Kim et al., 2005;Chen et al., 2006). The A-current in this region regulates the back-propagating action potential and synaptic integration (Hoffman et al., 1997). Therefore, Kv4.2 channels are critical regulators of postsynaptic excitability, and aberrant function or loss of Kv4.2 channels is likely to facilitate hyperexcitability and potentially seizure initiation and propagation in the hippocampus.Alterations in the Kv4.2 channel have been demonstrated in animal models of epilepsy. A decrease in Kv4.2 mRNA levels was found in rat hippocampus following pentylenetetrazol-induced seizures 1741-1751, 2009 doi: 10.1111/j.1528-1167.2009.02086.x FULL-LENGTH ORIGINAL RESEARCH1741 hippocampal excitability and a decrease in seizure threshold, there was a marked decrease in the expression of Kv4.2 channel subunits (Castro et al., 2001). Furthermore, in a rodent model of limbic seizures induced by pilocarpine, there was a significant decrease in Kv4...

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