Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration

Abstract

(1) Background: As membrane channels contribute to different cell functions, understanding the underlying mechanisms becomes extremely important. A large number of neuronal channels have been investigated, however, less studied are the channels expressed in the glia population, particularly in microglia. In the present study, we focused on the function of the Kv1.3, Kv1.5 and Kir2.1 potassium channels expressed in both BV2 cells and primary microglia cultures, which may impact the cellular migration process. (2) Methods: Using an immunocytochemical approach, we were able to show the presence of the investigated channels in BV2 microglial cells, record their currents using a patch clamp and their role in cell migration using the scratch assay. The migration of the primary microglial cells in culture was assessed using cell culture inserts. (3) Results: By blocking each potassium channel, we showed that Kv1.3 and Kir2.1 but not Kv1.5 are essential for BV2 cell migration. Further, primary microglial cultures were obtained from a line of transgenic CX3CR1-eGFP mice that express fluorescent labeled microglia. The mice were subjected to a spared nerve injury model of pain and we found that microglia motility in an 8 µm insert was reduced 2 days after spared nerve injury (SNI) compared with sham conditions. Additional investigations showed a further impact on cell motility by specifically blocking Kv1.3 and Kir2.1 but not Kv1.5; (4) Conclusions: Our study highlights the importance of the Kv1.3 and Kir2.1 but not Kv1.5 potassium channels on microglia migration both in BV2 and primary cell cultures.

Keywords: microglial cells; migration; pain; potassium channels; spared nerve injury.

Figure 1

Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 (A), Kv1.5 (B), Kir2.1 (C) and the negative control with Alexa 568 (D). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.

Figure 2

Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV (A–C), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD (n = 3 independent experiments).

Figure 3

The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h (A) and the cell migration in control conditions (E) and after the inhibitor at t24h (F–H). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 (B,D), whereas blocking Kv1.5 has no effect on cellular migration (C). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p < 0.001, using the parametric t-test (n = 3 independent experiments).

Figure 4

BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h (A) and the cell migration after scrambled and silencing siRNA at t24h (B–D) and at t48h (E–G). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h (H) and at t48h (I). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p < 0.001, using the parametric t-test (n = 3 independent experiments).

Figure 5

The migration of primary microglial cells through inserts with 8 µm pores. (A) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham (B–D) and SNI conditions (E–G). All the statistical analysis is represented as Mean ± SD, *: p < 0.05, ***: p < 0.001, using the nonparametric Mann–Whitney test (n = 3 independent experiments).

Figure 6

The experimental steps showing the inhibitory effect of potassium channels on microglial migration. (A) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. (B) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.