Developmental expression of Kv potassium channels at the axon initial segment of cultured hippocampal neurons

Abstract

Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation.

Figure 1. Developmental expression of potassium channels in cultured hippocampal neurons.

(A) Western blot of Kv1.2, Kv2.2 and Kv7.2 in hippocampal neurons cultured at high density (50,000/cm²) for different intervals (from 1 to 21 DIV) in control conditions. (B) Histograms show Kv1.2, Kv2.2 and Kv7.2 expression normalized to actin when quantified densitometry of Western blots. The data represent the mean ± SE of three independent experiments. Note the delayed onset of Kv1.2 expression as compared with that of Kv2.2 and Kv7.2. (C) Photomicrograph of hippocampal neurons cultured for 6 DIV and double immunostained for Kv7.2 (green) and MAP2 (red). Note the early expression of Kv7.2 in a single process emerging from the cell body (arrows). (D) Histogram shows the percentage (mean ± SE) of neurons expressing Kv7.2 at the AIS at different developmental stages in vitro. Scale bar = 16 µm.

Figure 2. Kv1.2 is concentrated at the AIS during axonal maturation in vitro.

Hippocampal neurons were grown for 1, 3, 6, 8, 10, 13, 15 and 18 days at low density (5,000/cm²), fixed in 4% PFA, and doubled stained with antibodies against Kv1.2 (green) and 14D4 antibodies (red) to identify the AIS. Note that 14D4 staining is detected at the moment of axon outgrowth in the nascent axon (A, B) and it is restricted to the AIS as the axon elongates (arrows). Confocal microscopy photomicrographs showing Kv1.2 immunostaining in hippocampal neurons cultured for up to 10 DIV (A–E). Staining is light and localized to the soma and neurites. After 10 DIV (F–H), intense Kv1.2 immunostaining is observed in the distal AIS. See Figure 3 for quantification. Scale bar = 18 µm.

Figure 3. Kv2.2 concentration at the AIS increases during axonal maturation in vitro.

Confocal microscopy photomicrographs showing representative hippocampal neurons cultured for 1, 3, 6, 8, 10, 13, 15, 18 and 20 days at low density (5,000/cm²), fixed in 4% PFA, and stained with antibodies against Kv2.2 (green) and VGSC (red). According to a previous study (Sánchez Ponce et al., 2008), VGSCs concentrate at the AIS (arrows) after 3 DIV. Note that moderate Kv2.2 immunostaining is localized homogeneously in the soma and proximal processes at all developmental stages in culture (A–C). After 14 DIV (D–F), Kv2.2 expression is evident in the axon in patches mainly distributed in the proximal region of the AIS. Histogram shows the percentage of neurons expressing Kv1.2 (G) and Kv2.2 (H) at the AIS at different developmental stages in vitro (the data represent the mean ± SE from three independent experiments). Scale bar = 18 µm (A–D) and 16 µm (E–F).

Figure 4. Lack of Kv1.2 and Kv2.2 colocalization at the AIS of cultured hippocampal neurons.

A–B and C–D: Pairs of representative confocal microscopy photomicrographs of hippocampal neurons cultured for 18 days, double immunostained for Kv1.2 (red) and Kv 2.2 (green), and counterstained with DAPI (A–D). Note that Kv1.2-expressing AISs (arrows) lack Kv2.2 immunostaining (Kv2.2) and vice versa (E). Histogram shows the proportion of neurons expressing Kv1.2, Kv2.2, neither or both at the same AIS at 18 DIV (the data represent the mean ± SE from three independent experiments). Scale bar = 25 µm.

Figure 5. AIS resistance to detergent extraction.

Hippocampal neurons cultured for 21 DIV were incubated for 15 min in a buffer containing 0.5% Triton X-100 before fixation (see Experimental methods) and analyzed by confocal microscopy. After detergent extraction, Kv1.2 (A) and Kv2.2 (D) were still present in the AIS, and they colocalized with the detergent resistant AIS markers 14D4 (B) and ankyrin G (E), respectively. Asterisks indicate the location of the neuronal soma. Scale bar = 12 µm.

Figure 6. AIS Kv channel expression is not dependent on the actin cytoskeleton.

Confocal microscopy photomicrographs show that Kv1.2 (A–F) and Kv2.2 (G–L) accumulation in the AIS is not affected by cytochalasin D. Hippocampal neurons were exposed to DMSO (control, A–C, G–I) or cytochalsin D (5 µM; D–F, J–L) from 15 to 17 DIV, double stained for 14D4 or ankyrin G (blue) and Kv1.2 (red, A–F) or Kv2.2 (red, G–L), and stained with Alexa 488 phalloidin to reveal F-actin. Note the presence of Kv1.2 and Kv2.2 at the AIS in both control and cytochalasin D-treated neurons. Scale bar = 25 µm (A–F) and 30 µm (G–L).

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.