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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.
Status epilepticus (SE) is a life-threatening condition defined as prolonged continuous seizures or intermittent seizures without recovery of consciousness. An episode of SE can be associated with the development of temporal lobe epilepsy (Leite et al. 2002). In several convulsant-induced models of limbic epilepsy (kainate and pilocarpine) in rodents, a prolonged episode of SE (90–120 min) is required for the development of epilepsy (Lemos and Cavalheiro 1995; Loscher 2002). A number of molecular and biochemical alterations likely occur in the limbic structures during prolonged SE. Insights into these alterations may provide novel targets for therapeutic intervention in SE.
The ERK 1/2 pathway regulates a broad range of target molecules through proline-directed serine/threonine phosphorylation (Kennelly and Krebs 1991). The ERK pathway downstream effector molecules in physiologic and pathologic conditions in the nervous system are currently being investigated (Thomas and Huganir 2004). The voltage-dependent K+ channel α-subunit, Kv4.2, is one of the substrates for ERK in hippocampus (Adams et al. 2000). Kv4.2 proteins localize to the somatodendritic regions of hippocampal neurons and contribute to the pore-forming regions of channels that express a transient, rapidly-activating K+ current (A-current) (Baldwin et al. 1991; Sheng et al. 1992; Maletic-Savatic et al. 1995; Martina et al. 1998; Serodio and Rudy 1998). The A-current attenuates action potential initiation and back-propagating action potentials (B-APs) and reduces excitatory synaptic events in CA1 dendrites, thereby modulating neuronal excitability (Hoffman et al. 1997; Martina et al. 1998; Migliore et al. 1999; Johnston et al. 2000; Cai et al. 2004). Kv4.2 knockout mice have loss of the A-current and an increase in the B-APs in hippocampal CA1 pyramidal cell dendrites (Chen et al. 2006), which suggests that the Kv4.2 channel is the major contributor to the A-current in this region. It has previously been shown that inhibition of ERK activation causes a hyperpolarizing shift in the voltage dependence of activation of the A-current in CA1 dendrites (Watanabe et al. 2002). Activation of the upstream regulators of the ERK pathway, cAMP-dependent protein kinase (PKA) and protein kinase C leads to down-regulation of the A-current in CA1 dendrites (Hoffman and Johnston 1999). ERK pathway modulation of the A-current in hippocampal CA1 dendrites is thought to be due to direct phosphorylation of Kv4.2 channel subunits.
Relatively little is known about the targets of the ERK pathway during acute seizures and SE. In the studies presented here, we found an increase in ERK phosphorylation of Kv4.2 channels during SE. These changes were evident at a synaptosomal level. Furthermore, total levels of Kv4.2 proteins were decreased within the synaptosomal and surface membrane subcellular compartments, while Kv4.2 levels were unchanged at a whole-cell level. We predict that increased ERK phosphorylation of Kv4.2 and decreased levels of Kv4.2 channels in the post-synaptic membrane of hippocampal neurons would contribute to hyperexcitability during SE.
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- Materials and methods
The findings presented here suggest that there are dynamic alterations in Kv4.2 channel post-translational modifications and post-synaptic localization during SE. Furthermore, we demonstrate that ERK activation during SE couples to phosphorylation of Kv4.2 in the hippocampus. The increases in ERK-phosphorylated Kv4.2 are reflected in the synaptosomal cellular compartment, which is expected to functionally down-regulate the A-current. An additional finding in our studies is that the pool of Kv4.2 channels is reduced in the synaptosomal and surface membrane subcellular compartments after prolonged SE. Given the critical role of Kv4.2 channels and its underlying A-current in hippocampal dendrites, we predict that the changes in Kv4.2 described here are likely to contribute to hyperexcitability and potentially recurrent seizures associated with SE.
The coupling of ERK activation to Kv4.2 phosphorylation during SE is predicted to have a significant impact on excitability in hippocampal neurons. ERK pathway activation leads to an increase in the amplitude of the B-APs in CA1 pyramidal cell dendrites, which is hypothesized to be via direct phosphorylation of Kv4.2 by ERK (Yuan et al. 2002). Indeed, studies in vitro using expression of phospho-site mutants at the mapped ERK sites within Kv4.2 compared with wildtype Kv4.2 channels in Xenopus oocytes resulted in functional alterations in Kv4.2 currents (Schrader et al. 2006). Phospho-mimetic mutation of the ERK sites resulted in an overall down-regulation of Kv4.2 currents, a depolarizing shift in channel activation, and slower recovery from inactivation compared with wildtype Kv4.2 currents. These findings were comparable with those described for the native A-current in hippocampal CA1 dendrites, where ERK pathway inhibition led to a hyperpolarizing shift in the activation curve for these currents (Watanabe et al. 2002). Work in the oocyte expression system also revealed differential functional effects of the three ERK sites within Kv4.2. Notably, phospho-mimetic mutation at the threonine 602 and 607 sites led to functional effects similar to that seen when all three sites were mutated to aspartate to mimic phosphorylation. Interestingly, phospho-mimetic mutation of the serine 616 site alone had the opposite effect. However, in the presence of phosphorylation at all three sites the effect of the threonine 602 and 607 sites seemed to override the functional effects of the serine 616 site and was similar to that identified when all three sites were mutated to aspartate (Schrader et al. 2006). Interestingly, our results using antibodies generated against the individual ERK phosphorylation sites within Kv4.2, revealed increases at all three ERK phosphorylation sites (greatest at threonine 602); thus, based on the results described above we predict that down-regulation of the A-current would be the predominant effect.
The mechanism underlying the decrease in Kv4.2 channels in the synaptosomal compartment in hippocampus following SE is currently undefined. Given that this change occurred relatively early following kainate-induced seizures and that Kv4.2 levels were unchanged in total cellular lysates and membranes, we hypothesize that there is altered trafficking or local translation of Kv4.2 channels during prolonged SE rather than a change in genomic transcriptional regulation of Kv4.2. Rapid alterations in GABAA receptor subunit trafficking have been reported in models of SE (Goodkin et al. 2005, 2007). GABAA receptors are regulated by PKA and endocytosis is clathrin dependent (Kittler and Moss 2003; Terunuma et al. 2008). Recent work from Kim et al. (2007) described clathrin-dependent internalization of Kv4.2 channels in cultured hippocampal neurons during glycine-induced long-term potentiation. In this model, Kv4.2 internalization was associated with increased synaptic plasticity and dendritic excitability. It is possible that the alterations in Kv4.2 synaptosomal and surface expression during SE reflect alterations in clathrin-mediated endocytosis and subsequent targeting for degradation.
While A-current regulation is best characterized in hippocampal area CA1, Kv4.2 channel proteins are localized to dendritic fields within area CA3 and dentate gyrus (Maletic-Savatic et al. 1995; Rhodes et al. 2004), where they may contribute to the A-current and regulate dendritic excitability in these regions. Our findings from whole-cell membrane preparations obtained from subdissected hippocampus reveal that increased ERK activation and phosphorylation of Kv4.2 are evident in each of these regions. The changes in synaptosomal levels of ERK-phosphorylated Kv4.2 and total Kv4.2 channels were evident in preparations harvested from whole hippocampus. The evaluation of whether there are subfield-selective changes in the regulation of synaptosomal Kv4.2 is a potentially important area for future investigations.
Alterations in Kv4.2 regulation and dendritic excitability have been demonstrated in epileptic animals (Bernard et al. 2004). There was a decrease in Kv4.2 protein and mRNA expression in hippocampal area CA1 from epileptic animals (Bernard et al. 2004). This study also revealed an increase in ERK phosphorylation of Kv4.2 in area CA1 from epileptic animals. Parallel physiology studies in the epileptic animals revealed an increase in the amplitude of the B-AP in the CA1 dendrites compared with controls. The alterations in Kv4.2 in the epileptic animals were thought to underlie this finding. In both the pilocarpine and kainate chemoconvulsant models of epilepsy, a prolonged episode of SE is required for the development of long-term, spontaneously recurring seizures. Thus, alterations in Kv4.2 channel regulation and expression are evident at both early and late time points of epileptogenesis in chemoconvulsant models. However, during chronic epilepsy with decreased Kv4.2 mRNA levels in area CA1 of hippocampus it appears that there is a change in Kv4.2 transcriptional regulation or mRNA processing. During SE, Kv4.2 mRNA levels are not significantly altered (unpublished data). Thus, the mechanisms involved in Kv4.2 expression may be different at the early and late time points of epileptogenesis.
We were interested in determining whether Kv4.2 represents a molecular locus for the convergence of multiple signaling pathways involved in the regulation of hippocampal excitability. We have previously mapped phosphorylation sites within Kv4.2 for PKA (Anderson et al. 2000). Thus, we evaluated PKA activation and phosphorylation of Kv4.2 channels during SE. However, our results suggest that while the PKA pathway is activated during SE, it does not appear to couple to direct PKA phosphorylation of Kv4.2 channels. It is possible that PKA couples to upstream activators of ERK in SE through B-Raf activation (Sweatt 2001; Rueda et al. 2002) and thus indirectly regulates the phosphorylation of Kv4.2 through ERK. Like the ERK pathway, there are many other candidate effector molecules for PKA in SE.
In our studies correlating behavioral with electrographic seizure activity, we found a significant discrepancy between the early and late electrographic and behavioral seizure events. The onset of electrographic seizure activity and SE occurred significantly earlier than the onset of the behavioral seizures and SE parameters (Racine scale). These findings were taken into account when investigating the time course of ERK activation and Kv4.2 phosphorylation following kainate administration. The lack of correlation of the behavioral and electrographic seizures should be a consideration when evaluating SE-induced molecular and physiological events.
In the time course studies, onset of non-convulsive electrographic seizures and SE correlated with the time course of the biochemical changes. ERK activation was significantly increased in all areas of the hippocampus by 30 min after kainate administration, which was after the onset of electrographic seizures but before the development of behavioral seizures and SE. An appreciable increase in ERK triply-phosphorylated Kv4.2 was not evident until the onset of electrographic SE (45 min after kainate) in CA1 and dentate gyrus. However, in CA3 significant increases in phospho-Kv4.2 were not evident until the later behavioral SE time point (180 min). The molecular basis of the lack of temporal correlation of ERK activation and phosphorylation of Kv4.2 is not understood at this time. Future investigations will be directed at this question.
A number of previous studies have suggested that there are modifications in ERK pathway signaling early following convulsant stimulation (Berkeley et al. 2002; Otani et al. 2003). However, the downstream molecular targets of the ERK pathway in SE models are not well defined. While there are a number of potential targets for ERK in hippocampus, our studies provide evidence for Kv4.2 as an ERK pathway target in the hippocampus during kainate-induced SE. Through phosphorylation of Kv4.2, our findings reveal a direct mechanism whereby the ERK cascade could contribute to the hyperexcitability of hippocampal neurons during SE. Thus, therapeutic interventions targeting the mechanisms defined here may prove useful during acute seizures and SE.