Altered phosphorylation and localization of the A-type channel, Kv4.2 in status epilepticus

The Cain Foundation Laboratories, Department of Pediatrics, Houston, Texas, USA.
Journal of Neurochemistry (Impact Factor: 4.28). 06/2008; 106(4):1929-40. DOI: 10.1111/j.1471-4159.2008.05508.x
Source: PubMed


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.

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    • "All samples were then stored at -80°C until used. Hippocampi were homogenized in ice-cold homogenization buffer (0.32 M sucrose, 1 mM EDTA, 5 mM Hepes) containing protease inhibitor cocktail (Sigma, USA) and processed for western blotting as previously described (Lugo et al., 2008). Through this procedure we produced total homogenate samples and crude synaptosomes. "
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    • "All samples were stored at −80°C until used. Hippocampi were homogenized in ice-cold homogenization buffer (100 mM Tris-HCl, pH 7.4, 0.32 M sucrose, 1 mM EDTA, 5 mM Hepes) containing protease inhibitor cocktail (Roche, Alameda, CA, USA) and processed for western blotting as previously described [31]. The protein concentration was determined using the Bradford Protein Assay (Bio Rad, Hercules, CA, USA). "
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    • "This effectively reduces the number of functional dendritic ion channels, even though the total HCN1 channel protein content remains unchanged. Similar alterations in ion channel trafficking to the surface membrane post-SE have been described for A-type K + channels and GABA A -receptor subunits, and thus may be a common theme in acquired channelopathy (Goodkin et al., 2008; Lugo et al., 2008; Terunuma et al., 2008). These latter examples are phosphorylation dependent , although such a mechanism has not yet been established for altered trafficking of HCN1 channels. "
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