Input-Specific Immunolocalization of Differentially Phosphorylated Kv4.2 in the Mouse Brain

Abstract

Voltage-gated A-type potassium channels such as Kv4.2 regulate generation of action potentials and are localized abundantly in the hippocampus and striatum. Phosphorylation consensus sites for various kinases exist within the sequence of the potassium channel subunit Kv4.2, including consensus sites for extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK), protein kinase A (PKA), protein kinase C (PKC), and calcium/calmodulin-dependent kinase II (CaMKII), and kinase assays have shown that particular amino acids of the consensus sites are bonafide phosphorylation sites in vitro. We have developed antibodies recognizing Kv4.2 triply phosphorylated at the three ERK sites as well as two antibodies recognizing singly phosphorylated Kv4.2 channels at the PKA sites (one amino-terminal and one carboxy-terminal). In the present study, we report the development of reliable immunohistochemistry protocols to study the localization of these phosphorylated versions of Kv4.2, as well as total Kv4.2 in the mouse brain. A general description of the areas highlighted by these antibodies includes the hippocampus, amygdala, cortex, and cerebellum. Such areas display robust synaptic plasticity and have been implicated in spatial, associative, and motor learning. Interestingly, in the hippocampus, the antibodies to differentially phosphorylated Kv4.2 channels localize to specific afferent pathways, indicating that the Kv4.2 phosphorylation state may be input specific. For example, the stratum lacunosum moleculare, which receives inputs from the entorhinal cortex via the perforant pathway, displays relatively little ERK-phosphorylated Kv4.2 or PKA carboxy-terminal-phosphorylated Kv4.2. However, this same layer is highlighted by antibodies that recognize Kv4.2 that has been phosphorylated by PKA at the amino terminus. Similarly, of the three antibodies tested, the soma of CA3 neurons are primarily recognized by the ERK triply phosphorylated Kv4.2 antibody, and the mossy fiber inputs to CA3 are primarily recognized by the carboxy-terminal PKA-phosphorylated Kv4.2. This differential phosphorylation is particularly interesting in two contexts. First, phosphorylation may be serving as a mechanism for targeting. For example, the amino-terminal PKA phosphorylation may be acting as a tag for a discrete pool of Kv4.2 to enter stratum lacunosum moleculare. Second, as phosphorylation may regulate channel biophysical properties, differential phosphorylation of Kv4.2 in the dendrites of pyramidal neurons may confer unique biophysical properties upon particular dendritic input layers.

Differential Expression ofIAChannel Subunits Kv4.2 and Kv4.3 in Mouse Visual Cortical Neurons and Synapses

“…High density of Kv4.2 and Kv4.3 subunits in thalamocortical input layers A striking finding of the present study is that the densities of the Kv4.2 and Kv4.3 subunits are highest in layer 1, the bottom of layer 2/3, in layer 4, and the layer 5/6 border. A similar laminar pattern was described in mouse somatosensory cortex, stained with antibodies against ERK (extracellular signal-regulated kinase)-phosphorylated Kv4.2 (Varga et al, 2000). The laminar pattern in mouse, however, differs from that in rat parietotemporal cortex, which shows extremely low Kv4.2 and Kv4.3 expression in layer 4 .…”

Kv4 potassium channels modulate hippocampal EPSP-spike potentiation and spatial memory in rats

“…Furthermore, in many areas of the brain, there is an overlap in the distribution of BmTX3 binding sites and the Kv4 subunits. In the hippocampal formation, only the combined distributions of Kv4.2 and Kv4.3 subunits are identical to those of the BmTX3 binding sites (Sheng et al 1992;Maletic-Savatic et al 1995;Serôdio et al 1996;Tsaur et al 1997;Serôdio and Rudy 1998;Varga et al 2000;Rhodes et al 2004;Jinno et al 2005;Menegola and Trimmer 2006;Vacher et al 2006). Finally, Kv4.2 and Kv4.3 are highly expressed in the hippocampal dentate gyrus (DG) (Tsaur et al 1997;Serôdio and Rudy 1998;Jinno et al 2005), a key structure to perform a radial maze task (Sutherland et al 1983;Jeltsch et al 2001).…”

Progressive Dendritic HCN Channelopathy during Epileptogenesis in the Rat Pilocarpine Model of Epilepsy

“…The hippocampi were then thawed in homogenization buffer (20 mM Tris-HCl at pH 7.5, 1 mM EGTA, 1 mM EDTA, 25 g/ml aprotonin, 25 g/ml leupeptin, 1 mM Na 4 P 2 O 7 , 500 M PMSF, 4 mM paranitrophenylphosphate, 1 mM sodium orthovanadate), and the CA1 region of the hippocampus was microdissected (Lubin et al, 2007). CA1 tissue samples were homogenized in 500 l of homogenatization buffer, normalized using a Bradford assay, and membranes prepared as described previously (Varga et al, 2000). The membrane samples from the experimental and control groups were loaded and run on a 10% acrylamide gel.…”