Implementation of screening strategy

As a first step to discover the sensor molecule(s) for galvanotaxis, we developed large-scale screening methods to identify ion channel genes that are important in galvanotaxis. We used the On-target plus siRNA human ion channel siRNA library. This library contains 381 siRNAs against genes coding human ion channels, pumps and transporters (Fig. 1, Supplementary Fig. 1). We transfected siRNA individually into telomerase immortalized human corneal epithelial cells (hTCEpi cells) using Lipofectamine 2000 reagent. Transfection efficiency was over 95% as judged from fluorescence of control oligo transfection (Supplementary Fig. 1c).

Figure 1: Large-scale RNAi screen for galvanotaxis phenotype. (a) hTCEpi cells transfected with siRNA from a library against human ion channels/pumps/transporters. (b) Polydimethylsiloxane (PDMS) stencil facilitated cell spotting in the galvanotaxis chamber. Cells, 48 h after transfection, were spotted onto galvanotaxis/electrotaxis chamber, pre-coated with FNC coating mixture, which could be guided by the stencil. (c) Multi-field video imaging to efficiently record cell migration of many types of cells in one experiment. After cells adhered to the culture dish, the stencil was removed. The chamber was covered with a coverslip. Direct current was applied. Cell migration was imaged with a time-lapse imaging system. (d) Knockdown of channels with the RNAi library revealed genes important for galvanotaxis. Graphs show migration directedness (cos θ) and migration speed from first screening of the whole library. Control is indicated by the blue line (cos θ=0.64). Migration speed was normalized to paired control (=1, indicated by blue line). (e) The screen identified genes critical for galvanotaxis. The y axis represents the z score of directedness (cos θ). Genes with z score >0.495 are highlighted in yellow, representing genes that after knockdown significantly increased galvanotaxis. Genes with z score <−0.7 are highlighted in red, representing genes that after knockdown significantly inhibited galvanotaxis. Cell numbers analysed for each conditions 35–69. EF=200 mV mm−1. Full size image

We used multi-spot seeding to screen for the galvanotaxis phenotype in large numbers of different types of cells. To increase screen efficiency, we developed stencils with multi-wells in which cells after different treatments could be seeded separately. Placing the stencil on the culture dish allowed us to simultaneously seed cells on spot arrays. Cells after transfection with different siRNA can therefore be seeded separately on each bottomless well without cross contamination. We used polydimethylsiloxane materials that adhere to the culture dish base with a water-tight seal that prevents well to well exchange of medium or cells. Our current galvanotaxis chamber allows up to 50 different treatments. At 48 h after transfection cells were trypsinized and seeded into the wells of the galvanotaxis chamber pre-coated with FNC Coating Mix. After cells adhere to the dish, the stencil can be lifted and removed (Fig. 1b,c). The cells were then exposed to EFs. On a motorized stage with multi-field video imaging, cells transfected with different siRNAs on up to 50 different spots can be video imaged at the same time. Galvanotactic migration was recorded with an inverted microscope for 30 min in a direct current EF of 200 mV mm−1, and quantitatively analysed using ImageJ. This method increased screening efficiency 50 times or more compared with traditional galvanotaxis experiments. Importantly, cells transfected with different siRNAs were processed and imaged at the same time in the same chamber together with the transfection control, minimizing batch to batch variation and significantly optimizing comparability of migration analyses.

RNAi screening identified genes important in galvanotaxis

We used the large-scale screening strategy to obtain galvanotaxis profiles after knockdown of individual ion channel subunits. We quantified directedness (cos θ) and migration speed using ImageJ software with MTrackJ and Chemotaxis Tool plugins (see Methods section). The directedness value quantifies how directionally the cells move in the field direction. Migration of a population of cells toward the cathode gives a directedness value larger than 0 and approaching 1, with a value of exactly 1 indicating a cell moved straight to the cathode. Migration of a population of cells towards the anode gives a directedness value smaller than 0 and approaching −1. Knockdown of some channels showed significant effects on both migration directedness and speed, while some affected migration directedness more than the speed, and some affected speed more than the directedness. Compilation of the directedness and speed data demonstrated the profiling of galvanotaxis after knockdown of individual ion channels in the library (Fig. 1d).

To identify which gene knockdowns showed significant effects on the migration speed and directedness, we set cutoff lines at 2.5% of the population distribution of both the directedness and migration values after knockdown. This analysis identified 35 gene knockdowns that showed significant effects on galvanotaxis. All except one affected migration speed or directedness separately, not both. After knockdown, 18 genes significantly affected directedness—KCNJ15, KCNAB2 and 7 others genes significantly decreased the directedness value, while knockdown of KCNJ5 or GABRG3 or any of other 6 genes significantly increased the directedness (Supplementary Fig. 2). Seventeen gene knockdowns significantly affected the migration speed—KCNA3, KCNA1 and seven other genes reduced the migration speed, while CLIC3, AQP3 and six other genes increased the speed. The one exception is ANO1; after knockdown, both migration speed and directedness increased significantly (Supplementary Fig. 2). In a few cases, there appeared to be distinctively separate roles for the same category of genes in regulation of speed and directedness. Knockdown of ligand-gated Cl− channels—GABRG3, GABRQ decreased the directedness without affecting migration speed, while the other family members GABRR2, GLRA1 and GLRA2 decreased the speed without significantly affecting the directedness (Supplementary Fig. 2). Voltage-gated K+ channels also showed similar separately regulated speed and directedness—KCNA7, KCNAB1, KCNAB2 reduced directedness, while KCNA1, KCNA3 decreased speed (Supplementary Fig. 2).

We performed a z score analysis which allows differentiation of more significantly different values from large samples (Fig. 1e). We set the cutoff value as a z score >0.495 or <−0.7, according to the upper and lower 2.5% of the distribution of the data, and this identified 18 genes. Knocking down nine candidates increased directedness, and knockdown of nine decreased directedness (Table 1). Knockdown of K+, Ca2+, Cl− and non-selective cation channels showed significant decrease or increase in galvanotaxis. The 18 genes identified include five K+ channels (KCNJ15, KCNJ5, KCNA7, KCNAB1 and KCNAB2), three that encode γ-subunits of voltage-gated Ca2+ channels (CACNGs—CACNG3, CACNG5 and CACNG8), two CLC Cl− channels, Ca2+-activated Cl− channel (ANO1), two ligand-gated Cl− channels (GABRG3 and GABRQ), two purinergic receptors (P2RX1 and P2RX5), water channel (MIP) and two other genes (ENSA and TNFAIP1).

Table 1 Genes which after knocking down caused impaired or enhanced galvanotaxis. Full size table

KCNJ15 specifically mediated the field sensing

To minimize possible interference of decreased speed on quantification of directedness, we grouped genes according to the effects on migration speed and directedness after knockdown. We chose to focus on genes that after knockdown showed significantly decreased directedness without significant effect on migration speed (rose-coloured part in Supplementary Fig. 2). KCNJ15 stood out; knockdown of KCNJ15, a gene encoding inwardly rectifying K+ channel Kir4.2, completely inhibited galvanotaxis while maintaining the same migration speed as non-target RNAi control (Table 1; Fig. 2c–e; Supplementary Video 1). Because this gene knockdown showed the most significant inhibition of directedness without affecting migration speed, we chose KCNJ15 for further study. Knockdown efficiency was confirmed by real-time quantitative PCR (qPCR) and western blot for mRNA and protein, respectively. Transfection of siRNA against KCNJ15 successfully reduced mRNA expression level by 80% (Supplementary Fig. 3a) and Kir4.2 protein level by 60% (Fig. 2a,b). Inwardly rectifying K+ channels, including KCNJ15/Kir4.2, are known to be important for the maintenance of resting membrane potential in various cells. We therefore measured the resting membrane potential of KCNJ15 knocked down cells. Resting membrane potential of KCNJ15 knocked down cells was significantly less negative (−38.98±0.66 mV; mean±s.e.m.) than that of control cells (−52.14±0.78 mV; Supplementary Fig. 4). To test whether other inward rectifying K+ channels may also participate in EF sensing, we tested KCNJ10/Kir4.1, which is also expressed in mouse corneal epithelial cells20. In the hTCEpi cells tested here, Kir4.1 appeared to localize exclusively in the perineuclear region (Supplementary Fig. 11, see below for details). Effective knocking down of KCNJ10 had significantly less effect on the membrane potential (−48.57±1.04 mV from −52.14±0.78 mV) than knocking down of KCNJ15 (Supplementary Figs 3b and 4), and also on galvanotaxis (cos θ=0.69±0.09 from 0.64±0.001) than knocking down of KCNJ15 (cos θ=0.12±0.11 from 0.64±0.001; Supplementary Table 1). KCNJ10/Kir4.1 perhaps plays a lesser role in both galvanotaxis and resting membrane potential maintenance than KCNJ15/Kir4.2 in the hTCEpi cells.

Figure 2: KCNJ15 knockdown specifically abolished galvanotaxis. (a,b) Efficient knockdown of KCNJ15 shown with Western blotting, red arrow pointing to a non-specific band. Kir4.2/GAPDH ratio is used to quantify the protein level. n=3. (c,d) Migration trajectories and quantification of directional migration (directedness values (cos θ)) demonstrated that KCNJ15 knockdown abolished galvanotaxis and cells completely lost migration direction in an EF. Black and red lines indicate trajectories of cells migrated toward cathode and anode side, respectively. n=100 cells for each group, confirmed in two other replicates. (e) KCNJ15 knockdown did not affect cell migration speed whether in an EF or not (compare the trajectories in c). There are no statistically significance between each group. n=100 cells for each group, confirmed in two other replicates. (f,g) Directional cell migration in scratch assay were identical between KCNJ15 knockdown and scrambled RNAi treatment. Wound closure is represented as % of open area. When the error bars are not seen, the bars are smaller than the symbols. Wound was made using a pipette tip. There are no statistically significance between two groups. n=3. Scale bar in f, 200 μm. (h) KCNJ15 knockdown abolished cathodal distribution of Akt-PH-EGFP, a reporter for PIP 3 localization. hTCEpi cells were transfected with siRNA and pcDNA3-Akt-PH-EGFP plasmid DNA. Fluorescence of Akt-PH-EGFP was recorded by fluorescence microscope. Arrow indicates PIP 3 accumulation in cathode-facing side of control cells. Scale bar, 50 μm. Cells were transfected with siRNA against KCNJ15 or control oligo, and incubated for 48 h. EF=200 mV mm−1. Statistical analyses were performed by Student’s t-test. Data represented as mean±s.e.m. *P<0.05; **P<0.01. NS, not significant. Full size image

To test the role of Kir4.2 with acute pharmacological treatment, we used Ba2+, a broad-range blocker for Kir channels. Ba2+ blocks inwardly rectifying K+ channels. Fifteen Kir channel-encoding genes (KCNJ1-6 and 8–16) have been identified in the human genome21, and Ba2+ inhibits them all. Ba2+ impaired galvanotaxis in a dose-dependent manner. Addition of BaCl 2 (100 or 500 μM) caused complete loss of galvanotaxis of the cells with directedness values returning to around 0, and significantly decreased migration speed (Fig. 3 and Supplementary Video 2 for 500 μM BaCl 2 , Supplementary Fig. 5 for 100 μM BaCl 2 ). Ba2+ inhibits Kir channels but not other types of K+ channels, such as voltage-gated K+ channels and Ca2+-activated K+ channels, at the concentration lower than millimolar order22.

Figure 3: Barium chloride treatment abolished galvanotaxis. (a) Cells treated with BaCl 2 lost galvanotaxis. Black and red lines indicate trajectories of cells migrated toward cathode and anode side, respectively. n=100 cells for each group, confirmed in two other replicates. (b) Directedness values (cos θ) confirm loss of directedness. n=100 cells for each group, confirmed in 2–3 other replicates. (c) BaCl 2 treatment significantly inhibited migration speed. n=100 cells for each group, confirmed in 2–3 other replicates. (d) BaCl 2 treatment prevented asymmetric accumulation of PIP 3 to the leading edge. hTCEpi cells were transfected with pcDNA3-Akt-PH-EGFP plasmid DNA. Fluorescence of Akt-PH-EGFP was recorded by fluorescence microscope. Arrow indicates PIP 3 accumulation in cathode-facing side of control cells. Scale bar, 50 μm. BaCl 2 was used at 500 μM. EF=200 mV mm−1. Statistical analysis was performed by the Student’s t-test. Data represented as mean±s.e.m. **P<0.01. Full size image

We then investigated the specificity of KCNJ15 in EF sensing. Cells after KCNJ15 knockdown lost directedness in an EF, but maintained the same migration speed as non-target siRNA control cells or cells without an EF. The role for KCNJ15 therefore appeared to be specific for directional sensing in an EF, not a general inhibition of cell motility (Fig. 2c–e). Migration trajectories of KCNJ15 knockdown cells are similar to those of no EF cells (both control oligo- and KCNJ15 siRNA-transfected cells). Cell migration in a monolayer scratch assay was identical in KCNJ15 knockdown and non-target RNAi control. KCNJ15 knockdown did not have any effect on wound closure, suggesting that the responsiveness of the cells to the directional cues (including injury, free edge and contact inhibition release) in this model remained the same (Fig. 2f). Several KCNJ genes are reported to be expressed in mouse corneal epithelial cells20,23. Knockdown of other KCNJ genes except KCNJ14 (encoding Kir2.4) had no effect on the directedness (Supplementary Table 1). The inhibitory effect of BaCl 2 was most likely through inhibition of Kir4.2 (KCNJ15). These results indicate that KCNJ15 knockdown specifically affected sensing of the field, not motility or directional migration in a monolayer scratch assay.

The effects of KCNJ15 knockdown on galvanotaxis at different EF strengths show the inhibition was complete up to 500 mV mm−1. Non-target control siRNA-transfected cells started to respond at 30 mV mm−1, and reached the maximum level at around 100 mV mm−1. BaCl 2 -treated cells showed the same loss of directedness in higher EF strength (Supplementary Fig. 6).

Knockdown of KCNJ15 prevented PIP 3 polarization

Next, we determined the distribution of PIP 3 , a cell polarization marker, in cells after KCNJ15 knockdown or Kir channel inhibition. Cells undergoing directional migration, including galvanotaxis, recruit PIP 3 to the leading edge8,24,25,26. We transfected hTCEpi cells with KCNJ15 siRNA followed by an expression construct of pleckstrin-homology domain of Akt fused with enhanced green fluorescence protein (Akt-PH-EGFP), or transfected with an Akt-PH-EGFP construct and treated with BaCl 2 . Akt-PH-EGFP reports PIP 3 localization. In an EF, Akt-PH-EGFP redistributed to the cathode-facing side of hTCEpi cells. Cathode-polarization of Akt-PH-EGFP however was not observed in KCNJ15 knockdown cells and BaCl 2 -treated cells (Figs 2h and 3d; Supplementary Tables 2 and 3).

KCNJ15/Kir4.2 is also required for anode galvanotaxis

In an EF, some types of cell migrate directionally to the anode, opposite to the direction of galvanotaxis of the corneal epithelial cells. To determine whether KCNJ15 is required for anode galvanotaxis we transfected KCNJ15 siRNA into two lines of anode-migrating cells. HaCaT cell (spontaneously immortalized human keratinocytes) and MDA-MB-231 cell (human breast adenocarcinoma line) migrated to the anode as shown by the negative directedness value (cos θ). Directional migration of both cell lines was lost after knockdown of KCNJ15 (Fig. 4). KNCJ15/Kir4.2 thus is essential to both cathodal and anodal galvanotaxis. KCNJ15 knockdown did not affect migration speed in HaCaT cells, as in hTCEpi cells. Knockdown of KCNJ15 in MDA-MB-231 cells reduced migration speed as in mouse embryonic fibroblasts27. These observations may suggest that KCNJ15 is specific in directional sensing in an EF; its involvement in regulating migration speed may be cell-type depend.

Figure 4: KCNJ15 knockdown abolished cathode as well as anode galvanotaxis. (a) hTCEpi cells migrate toward cathode, and HaCaT (human keratinocyte cells) and MDA-MB-231 (human breast cancer cells) cells migrate toward anode. Knockdown of KCNJ15 abolished directional migration in all three types of cells. At 48 h after transfection, cells were seeded onto galvanotaxis chamber. Positive directedness values indicate cathodal migration, whereas negative directedness value indicates anodal migration. (b) KCNJ15 knockdown did not have significant effects on migration speed. hTCEpi cells, HaCaT and MDA-MB-231 were transfected with siRNA against KCNJ15 or control oligo. Cell numbers analysed, 100 hTCEpi cells, 60 HaCaT cells and 80 MDA-MB-231 cells. Results confirmed in two separate experiments. Statistical analysis was performed by the Student’s t-test. Data represented as mean±s.e.m. *P<0.05. **P<0.01. EF=200 mV mm−1. NS, no significance. Full size image

We also determined the distribution of PIP 3 in anode-migrating HaCaT cells and MDA-MB-231 cells transiently transfected with Akt-PH-EGFP. No obvious polarized localization of Akt-PH-EGFP was observed in those anode-migrating cells (Supplementary Fig. 7).

Kir4.2 coupled with polyamines to sense the EF

To elucidate the mechanisms of KCNJ15/Kir4.2 in sensing an EF, we examined the effects on galvanotaxis of pore blocking the Kir channels. Kir channels do not possess a canonical voltage sensing domain and have unique features unlike voltage-gated K+ channels21. Kir channels allow K+ more easily to flow into the cells than out of the cells. Intracellular polyamines regulate inward rectification activities of Kir channels. Polyamines are small organic compounds that have two or more primary amino groups, therefore carrying positive charges at regularly spaced intervals. In mammalian cells, spermidine (SPD), spermine (SPM) and putrescine (PUT) are three major polyamines. SPM and SPD, which have +4 and +3 charges, respectively, have enough size and charge to block Kir channels, whereas PUT does not. Highly positively charged polyamines bind to negatively charged residues, for example, glutamate and aspartate, located at the channel pore region. Polyamine depletion altered the inward rectifying property of Kir channels; that is, K+ flow reversed to outward rather than inward28,29,30,31.

To test the role of polyamines (SPM/SPD) in galvanotaxis, we depleted intracellular polyamines by treating cells with polyamine analogue N1, N11-diethylnorspermine (DENSPM). Incubating cells with DENSPM, a potent activator of polyamine-catabolizing enzyme SPM/SPD acetyltransferase (SAT/SSAT), reduces intracellular SPM/SPD by catalysing the transacetylation reaction. Treatment with DENSPM completely abolished galvanotaxis (Supplementary Video 3; Fig. 5a–c). Cells migrated in random directions, as in KCNJ15 knockdown and blocker experiments. Migration trajectories of DENSPM treated cells showed random migration similar to RNAi and blocker experiments (Figs 2 and 3; Supplementary Fig. 5).

Figure 5: KCNJ15 couples with polyamines to sense extracellular EFs. (a,b) Intracellular polyamines are required for cells to sense extracellular EFs. Depletion of polyamines with DENSPM abolished galvanotaxis. Migration trajectories of control cells and DENSPM treated cells with or without EF. Red lines indicate trajectories of cells that migrated toward anode side. hTCEpi cells were treated with 25 μM DENSPM for 2 days. DENSPM activates SPM/SPD catabolizing enzyme SAT/SSAT which catabolizes SPM/SPD to N1-acetyl SPM/SPD and reduces intracellular polyamine. (c) Depletion of polyamines affected cell migration speed. (d,e) Increased intracellular polyamines significantly enhanced galvanotaxis of U251 cells (d) or HaCaT cells (e). Knockdown of KCNJ15 cancelled PUT-enhanced galvanotaxis. Cells were transfected with control oligo or KCNJ15 siRNA, and treated with or without PUT (100 μM) for 2 days. (f) Lentivirus-mediated expression of WT and E157N Kir4.2 proteins. hTCEpi cells transduced with lentivirus. The Expression of WT and polyamine-binding defective mutant (E157N) Kir4.2 proteins was confirmed by western blotting. (g,h) Expression of polyamine-binding defective mutant of KCNJ15 (E157N) significantly decreased directedness but had little effect on cell motility. hTCEpi cells were infected with recombinant lentivirus and incubated for 2 days. Directedness and speed were evaluated. (i,j) An applied EF-induced asymmetry of intracellular polyamines. Representative image of the polyamine staining (i). Scale bar in i, 50 μm. Intensities of polyamines staining in cathode-facing side were divided by those in anode-facing side (right side divided by left side in no EF cells) (j), Polyamine distribution in response to EF. hTCEpi cells were subjected to EF (200 mV mm−1) for 0, 10, 30 or 60 min. Intracellular polyamines were stained with anti-polyamine antibody. Arrows in i indicate polyamine accumulation in cathode-facing side of hTCEpi cells. Statistics: b and c: n=100 cells for each group, confirmed in three independent experiments. d: n=50, e: n=120, g and h: n=120, j: n=16–23. All confirmed in two to three separate experiments. EF=200 mV mm−1. Statistical analyses were performed by Student’s t-test (b–e,g,h), or analysis of variance followed by Student’s t-test (j). Data represented as mean±s.e.m. *P<0.05. **P<0.01. NS, no significance. Full size image

We then increased the intracellular concentration of polyamines by incubating HaCaT and U251 cells with PUT, which is an important precursor of SPM/SPD synthesis. Treatment with PUT increases intracellular SPM/SPD concentrations27. PUT treatment significantly enhanced directedness of both HaCaT and U251 cells (Fig. 5d,e). The stimulatory effect of PUT on galvanotaxis was abolished by knocking down of KCNJ15 in both HaCaT cells (anode migrating) and U251 (cathode migrating) cells. Importantly, knockdown of KCNJ15 completely diminished PUT-induced enhancement of galvanotaxis (Fig. 5d,e).

To determine the role of interaction between channel protein and polyamines in galvanotaxis, we expressed polyamine-binding defective KCNJ15. Kir channels function as a tetramer in the plasma membrane. The pore region of Kir channels has a negatively charged amino-acid residue (corresponding to E157 of human Kir4.2), which interacts with polyamines. Mutation of this residue increased outward current32. Coexpression of wild type (WT) and mutant increased outward current that was intermediate between WT and mutant homo-tetramer32. Substitution of Glu-157 with Asn (E157N) resulted in complete loss of the inward rectification property of Kir4.2 (ref. 27). The mutant channels, if expressed in the cells, would act as ‘dominant negative’. We produced recombinant lentivirus to express mutant KCNJ15 (E157N) and infected hTCEpi cells. Expression of E157N in hTCEpi cells decreased directedness (cos θ) but had little effect on cell motility (Fig. 5f–h). We further determined if expression of E157N affects PIP 3 polarization in hTCEpi cells. We infected recombinant lentivirus to express WT or E157N, then transfected the expression construct of Akt-PH-EGFP. In WT expressing cells, about 38% of cells showed cathodal polarization of PIP 3 (Akt-PH-EGFP) within 30 min after EF application. On the contrary, in E157N-expressing cells, only ∼24% of cells showed cathodal polarization of PIP 3 , which was significantly lower than WT expressing cells (P<0.01; Supplementary Fig. 8). These observations suggest that the interaction between Kir4.2 protein with intracellular polyamines is required to sense extracellular EFs, and modulation of the inward rectification properties of Kir4.2 by polyamines might be an important factor for polarization during galvatanoxis.

EF-induced asymmetrical distribution of polyamines

To test if EFs cause asymmetrical distribution of these highly positively charged molecules, we applied an EF to hTCEpi cells and fixed them after different times in the EF (10, 30, 60 min and no EF control). The cells were stained with anti-polyamine antibody. SPM and SPD staining was much higher at the cathode-facing side than that at the anode-facing side (Fig. 5i,j). We measured the intensity of polyamine staining at both sides (cathode and anode, or right and left in no EF control) and calculated the cathode/anode or right/left ratios. Polyamines were accumulated at the cathode-facing side in response to EF application, and accumulation was increased in a time-dependent manner. Intracellular polyamines also accumulated at the cathode-facing side in response to EF in anode-migrating HaCaT and MDA-MB-231 cells (Supplementary Fig. 9)

We then tested if EFs cause asymmetrical distribution of Kir4.2 protein in hTCEpi cells. We applied an EF to hTCEpi cells and the cells were fixed and stained with anti-Kir4.2 antibody. F actin was visualized by using Alexa555-conjugated phalloidin. Kir4.2 protein was localized at the intracellular region (mostly perinuclear region), and the membrane expanding region (many dispersed dots in those regions). After application of EF, Kir4.2 protein signal was still observed in both cathode-facing and anode-facing sides, and intracellular region without obvious polarization (Supplementary Fig. 10).

Corneal epithelial cells express Kir4.1, which is encoded by KCNJ10 (ref. 20) and has 62% amino-acid identity to Kir4.2. Immunostaining showed a different subcellular distribution of Kir4.1 from that of Kir4.2. Kir4.1 proteins were mainly localized at the intracellular perinuclear region and not expressed in the membrane expansion region (Supplementary Fig. 11). In addition, knocking down of KCNJ10 had little effect on the directedness (cos θ; Supplementary Table 1). Consistently, membrane potential of KCNJ10 knocked down cells was similar to that of control cells (Supplementary Fig. 4). These observations suggest that the functional contribution of KCNJ10/Kir4.1 to maintenance of resting membrane potential and galvanotaxis is significantly smaller than that of KCNJ15/Kir4.2.