Input-specific immunolocalization of differentially phosphorylated Kv4.2 in the mouse brain

Voltage-gated A-type potassium channels such as Kv4.2 regulate generation o f action potentials and are localized abundantly in the hippocampus and str iatum. 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/ca lmodulin-dependent kinase II (CaMKII), and kinase assays have shown that pa rticular amino acids of the consensus sites are bonafide phosphorylation si tes in vitro. We have developed antibodies recognizing Kv4.2 triply phospho rylated at the three ERK sites as well as two antibodies recognizing singly phosphorylated Kv4.2 channels at the PKA sites tone amino-terminal and one carboxy-terminal). In the present study, we report the development of reli able immunohistochemistry protocols to study the localization of these phos phorylated 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 t he 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 differ entially phosphorylated Kv4.2 channels localize to specific afferent pathwa ys, indicating that the Kv4.2 phosphorylation state may be input specific. For example, the stratum lacunosum moleculare, which receives inputs from t he entorhinal cortex via the perforant pathway, displays relatively little ERK-phosphorylated Kv4.2 or PKA, carboxy-terminal-phosphorylated Kv4.2. How ever, this same layer is highlighted by antibodies that recognize Kv4.2 tha t has been phosphorylated by PKA at the amino terminus. Similarly, of the t hree antibodies tested, the soma of CA3 neurons are primarily recognized by the ERK triply phosphorylated Kv4.2 antibody, and the messy fiber inputs t o CA3 are primarily recognized by the carboxy-terminal PKA-phosphorylated K v4.2. This differential phosphorylation is particularly interesting in two contexts. First, phosphorylation may be serving as a mechanism for targetin g. For example, the amino-terminal PKA phosphorylation may be acting as a t ag for a discrete pool of Kv4.2 to enter stratum lacunosum moleculare. Seco nd, as phosphorylation may regulate channel biophysical properties, differe ntial phosphorylation of Kv4.2 in the dendrites of pyramidal neurons may co nfer unique biophysical properties upon particular dendritic input layers.