Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons
Figure 7
AIS Kv channel concentration is dependent on ankyrin G.
Confocal microscopy photomicrographs show that interference RNAs against ankyrin G impair the concentration of Kv1.2 and Kv2.2 at the AIS. Before plating, hippocampal neurons were nucleofected with plasmids expressing scrambled shRNA (A–C, G–I) or ankyrin G shRNA (D–F, J–L), and subsequently cultured until 18 DIV. The neurons were then double stained with antibodies against ankyrin G or pIκBα (14D4 blue) and Kv1.2 or Kv2.2 (red). Nucleofected neurons were identified by GFP fluorescence. Ankyrin G, the protein recognized by 14D4 antibodies, Kv1.2 and Kv2.2 concentrated in the AIS of neurons expressing scrambled shRNA plasmids (A and G) and in non-nucleofected neurons (arrowhead in D–F). However, in the absence of ankyrin G in neurons nucleofected with ankyrin G shRNA (D–F, J–L), no Kv1.2 or Kv2.2 immunostaining was observed at the AIS. Note that patches of Kv2.2 immunostaining in the cell somata were not affected by ankyrin G interference (K). Scale bar = 25 µm. Arrows indicate AISs and asterisks indicate the somas of non-nucleofected neurons.