The Role of Kv1.2 Channel in Electrotaxis Cell Migration

Abstract

Voltage-gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF-induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS-7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time-lapse imaging analysis showed that EF-induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS-7 cells to EF stimulation more than their motility. Immunocytochemistry and pull-down analyses demonstrated association of Kv1.2 channels with actin-binding protein cortactin and its re-localization to the cathode-facing membrane at EF stimulation, which confirms the mechanism of EF-induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF-induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy, and various degenerative syndromes.

Figure 1

Kv1.2 clone and expression. Mouse K⁺ channel Kv1.2 was cloned into pcDNA3.1 plasmid and three stable Kv1.2 transfected COS‐7 cell lines were generated: Kv1.2‐A, Kv1.2‐B, and Kv1.2‐C. A,B: western blot showed different expression of Kv1.2 in three clones, and expression intensity analysis confirmed that Kv1.2‐C clone had the highest Kv1.2 expression in comparison to WT cells, Kv1.2‐A and Kv1.2‐B clones. C,D: quantitative PCR analysis of Kv1.2 expression in the three Kv1.2 clones confirmed the results in Western blot, comparing to two different house‐keeping genes GAPDH and HPRT, respectively. Results are presented as means ± SE; n ≥ 3. *P < 0.05.

Figure 2

Currents and membrane potential of Kv1.2 Clones recorded in conventional whole‐cell configuration. A–C: mean current densities (pA.pF⁻¹) comparison of transmembrane outward currents in COS‐7 WT cells (n = 8), against low (A: Kv1.2‐A, n = 6), medium (B: Kv1.2‐B, n = 5), and high (C: Kv1.2‐C, n = 11) level of Kv1.2 channel expression, respectively. D: diagram of mean membrane potential (mV) of WT COS‐7 cells compared to low, medium and high level of Kv1.2 channel expression (Kv1.2‐A, B, and C). *P < 0.005, ** P < 0.003, ***P < 0.0003. Mean ± SE.

Figure 3

Overexpression of Kv1.2 enhanced electrotaxis of COS‐7 cells. A–I: time lapse imaging was conducted for WT and Kv1.2‐C cells in the absence or presence of 100 mV/mm EF for 4 h, and cell migration behaviors were analyzed with ImageJ software. Cell migration trajectories were accumulated and composed with starting points at the same origin point (0,0) on axes (A,D,G). Middle (B,E,H), and right (C,F,I) columns represent the first and last frame of the real‐time imaging, respectively. The black lines indicate the cell migration trajectories; “–” and “+” indicate the cathode and anode of the electric stimulation, respectively. J–K: Kv1.2‐C clone and WT cells were treated with EF from 0 to 300 mV/mm and their migration directedness (J) and speed (K) were summarized. Results are presented as means ± SE; n ≥ 3, *P < 0.005.

Figure 4

Kv1.2 inhibition diminishes electrotaxis in COS‐7 cells. A: exemplar traces of transmembrane outward currents in Kv1.2‐C clone recorded in conventional whole‐cell configuration generated by 80 ms voltage steps from −120 to +80 mV, in incremental steps of 10 mV from a holding potential of −70 mV in Control (upper part) in the presence of ChTX 0.5 μM (middle part). ChTX‐sensitive current (lower part in A) was obtained by mathematic subtraction of transmembrane current in the presence of ChTX 0.5 μM from transmembrane current in the Control. B: mean current densities (pA.pF‐1) of WT COS‐7 cells (upper part, n = 11) and COS‐7 cells stably expressing Kv1.2 channels (lower part, n = 11) were recorded in conventional whole‐cell configuration voltage step protocol (see above A); ● represents control, ▴ in the presence of ChTX 0.5 μM, and ○ ChTX‐sensitive current (Control ChTX 0.5 μM). Kv1.2‐C cells were treated with either EFs (100 mV/mm) only or EFs plus ChTX (0.5 μM) and the migration directedness (C) and speed (D) were analyzed, respectively. E,F: composing migration trajectories of Kv1.2‐C cells with EF (100 mV/mm) and EF + ChTX (0.5 μM) by pointing all the trajectories starting from the origin point on axes (0,0), respectively. Results are presented as means ± SE; n ≥ 3. *P < 0.05.

Figure 5

EF increased interaction between Kv1.2 channels and cortactin. The Protein A/G Argose‐Kv1.2 antibody immunoprecipitation (IP) pull‐down assay with indicated Kv1.2‐C cell lyses using cortactin antibody. The blot results show that EF increases the binding between Kv1.2 and cortactin. However, the interaction in WT cells was not observed even after EF stimulation (A and B). Additionally, neither overexpression of Kv1.2 nor EF treatment changed cortactin expression (A and C). Results are presented as means ± SE; n ≥ 3. *P < 0.05.

Figure 6

EF polarized Kv1.2 and cortactin co‐localization at the cathode‐facing membrane of migration cells. A–F: Confocal immunocytochemistry staining of Kv1.2 (green) and cortactin (red) on Kv1.2‐C cells in the absence or presence of EF (150 mV/mm) treatment, respectively. A',B'; D',E': the fluorescent profile of Kv1.2 and cortactin across the cell paralleling to EF vector were analysed accordingly with ImageJ software. White line indicates the analysed area of the fluorescence profile; A',B' show the fluorescence intensity profiles of the cells without EF, and D',E' with 150 mV/mm EFs for 2 h. G: co‐localization of Kv1.2 and cortactin at the cathode‐facing membrane on Kv1.2‐C clone in the absence or presence of EF stimulation. The white rectangles on C and F are the analyzed areas. Means ± SE; n ≥ 3. *P < 0.05.

Figure 7

Schematic illustration of a hypothetical mechanism for Kv1.2 in electrotaxis. The most prominent function of Kv1.2 channel is to control the electrogenic transport of ions across the plasma membrane. It hyperpolarizes the cell membrane potential by elevating the extracellular K⁺ concentration. Cortactin is highly enriched in lamellipodia and enhances protrusion persistence, which potentially binds to F‐actin to regulate membrane dynamics and form adhesion nucleus through regulation of Arp2/3 and interaction with cadherin. The Kv1.2 can be suppressed by activated RhoA and the tyrosine phosphorylation at its C‐terminal which blocks its interaction to cortactin. When treated with EF, Kv1.2 channel may act as sensors responding to the electric signals, and subsequently activated Kv1.2 channel binds cortactin at the leading edge. The asymmetrical colocalization of Kv1.2 channel and cortactin at the cathode‐facing membrane of the migration cells enhances actin polymerization and protrusion thereby the directional migration is initiated.