Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium

RESEARCH ARTICLE Open Access Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium Julia K. Johansson 1 , Viivi I. Karema-Jokinen 1 , Satu Hakanen 2 , Antti Jylhä 1 , Hannu Uusitalo 1,3 , Maija Vihinen-Ranta 2 , Heli Skottman 1 , Teemu O. Ihalainen 1 and Soile Nymark 1* Abstract Background: Voltage-gated sodium (Na v ) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Na v channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Na v channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Na v channels in RPE both ex vivo and in vitro. Results: Our work demonstrates that several subtypes of Na v channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Na v 1.4, Na v 1.6, and Na v 1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Na v subtypes. Importantly, we show that the Na v channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Na v 1.4 and Na v 1.8 accumulate on phagosomes and that pharmacological inhibition of Na v channels as well as silencing the expression of Na v 1.4 with shRNA impairs the phagocytosis process. Conclusions: Taken together, our study shows that Na v channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis. Keywords: RPE, Ion channels, Na v , Patch clamp, Phagocytosis, Retina, Photoreceptors Introduction In the vertebrate eye, the retinal pigment epithelium (RPE) forms a barrier between the retina and the choroid [1–3]. Its cells are associated closely with photoreceptors: their apical sides surround the outer segments with long microvilli, and the basolateral sides are attached to Bruch’s membrane, an extracellular matrix separating the RPE from the choroid [3,4]. The RPE has many functions that are vital to retinal maintenance and vision, such as maintaining the visual cycle, secreting important growth factors, delivering nutrients to the photoreceptors from the bloodstream while removing metabolic end products, and absorbing scattered light [1,3]. Additionally, RPE maintains ionic homeostasis in the subretinal space [5] and sustains photoreceptor renewal by phagocytosing their shed outer segments [1,6]. Phagocytosis is highly essential for vision, and it is under strict diurnal control, initiated at light onset for rods and typically at light offset for cones [7,8]. This evolutionarily conserved molecular pathway is receptor mediated and precisely regulated; however, the exact signaling cascades are still not completely understood [9]. Recent studies imply the importance of specific ion channels in this process including the L-type calcium channels as © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: [email protected] 1 BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland Full list of author information is available at the end of the article Johansson et al. BMC Biology (2019) 17:63 https://doi.org/10.1186/s12915-019-0681-1 well as calcium-dependent potassium and chloride channels [10–12]. Since the first single-cell recordings from RPE in 1988 [13], a large variety of different ion channels have been identified in them [5]. Among these are several voltagegated calcium, potassium, and chloride channels. However, the identity of sodium conductive ion channels in RPE has remained elusive [5], even though the importance of sodium homeostasis to normal RPE function is acknowledged. Of the two main families of sodium channels, there is evidence of both epithelial Na + channels and voltage-gated Na + (Na v ) channels in RPE [5,14, 15,18,19]. However, electrophysiological data demonstrating their functionality is missing in mature RPE. More importantly, Na v channels that are characteristic of excitable cells have to date only been detected from cultured RPE. This has resulted in the interpretation that their expression is due to neuroepithelial differentiation that can occur in culture [5,20,21]. Here, we shed light on this crucial issue by demonstrating thepresenceofNa v channels both in cultured human embryonic stem cell (hESC)-derived RPE and freshly isolated mouse RPE. We show that Na v channels co-regulate photoreceptor outer segment (POS) phagocytosis. Our hypothesis is supported by a recent demonstration of the involvement of Na v channels in phagocytosis of mycobacteria by macrophages [22]. Our work provides evidence that Na v 1.8 accumulates with the phagosomal particles. Na v 1.4 also accumulates to phagosomes but displays localization to cell–cell junctions outside phagocytosis. Interestingly, selective Na v channel blockers significantly reduced this phagosomal translocation. Moreover, the selective blockers combined with the universal Na v blocker tetrodotoxin (TTX) reduced the total number of ingested POS particles by up to 41% while not affecting their binding. Reduction was also observed when the expression of Na v 1.4 was silenced with short hairpin RNA (shRNA). More generally, our observations add to the growing body of evidence that Na v channels play diverse roles in a variety of classically non-excitable cell types ranging from astrocytes and microglia to macrophages and cancer cells (for review, see [23]). Collectively, our results show that this epithelium is electrically more complex than was previously thought. Results Functional voltage-gated sodium channels are present in RPE derived from human embryonic stem cells We used whole-cell recordings from mature hESCderived RPE in K + free intracellular solution to observe transient inward currents elicited by a series of depolarizing voltage pulses after strong hyperpolarization to − 170 mV (Fig. 1c, n= 19). These recordings were performed from an intact monolayer (Fig. 1a, results summarized in Fig. 1j) in the presence and absence of a gapjunction antagonist (18α-glycyrrhetinic acid). Resembling currents, but with only a fraction of the amplitude, were occasionally identified in cells from freshly dissociated mature hESC-derived RPE (Fig. 1b, d, n= 6), that is the conventional configuration for RPE patch clamp recordings. The current resembled the Na v current characteristic of excitable cells: it had the typical current– voltage relationship (Fig. 1e) and showed fast activation and inactivation (Fig. 1i). The current was activated at about −50 mV and peaked at about −13 mV with a maximum amplitude of 330 ± 50 pA (mean ± SEM, n= 19). The average membrane capacitance was 28 ± 2 pF (n= 19), and the average current density was 13 ± 3 pA/ pF (n= 19). The average resting membrane potential, measured in the presence of K + -based intracellular solution was −47 ± 1 mV (mean ± SEM, n= 15). The inactivation time constant decayed exponentially with increasing command voltages, while the decay of the activation time constant was more shallow (Fig. 1i). The steady-state inactivation curve was determined by measuring the amplitude of a response to −10 mV test pulse following a series of prepulses (from −140 mV to −40 mV at 10 mV intervals). The normalized current amplitude was plotted against the prepulse voltage and fitted with the Boltzmann equation I=Imax VðÞ¼1=1þexp V−V1=2  =k  ð1Þ resulting in the half-inactivation voltage V 1/2 =−94 ± 1 mV (n=7)(Fig.1f). To investigate the time dependency of recovery from inactivation, we used a paired-pulse protocol (Fig. 1g). The current was recorded after a second depolarizing pulse given at increasing time intervals until it finally recovered to its full size. The second peak currents were subsequently normalized to theprepulsepeakcurrentand plottedagainstthetimebetweenthetwovoltagepulses (Fig. 1h). Our data was fitted with an exponential function, and the best fit yielded to τ=54±3ms(n=5). The presence of Na v currents was confirmed using the universal extracellular Na v channel blocker TTX. By comparing the responses elicited with a voltage step from −170 to −10 mV, it was evident that addition of 1μM TTX to the bath reduced the amplitude of the current to roughly one half of that recorded in the control extracellular solution (Fig. 2a, left). Thus, the recorded current was sensitive to TTX but required reasonably high concentrations. Furthermore, the sensitivity to TTX varied between the cells and in some cases even 10 μM TTX was not enough to block the current (Fig. 2a, left). The current was also sensitive to 2 mM QX-314, an intracellular Na v channel blocker added to the internal solution of the patch pipette that typically removed the current rapidly after breaking into the whole-cell configuration (Fig. 2a, right). Johansson et al. BMC Biology (2019) 17:63 Page 2 of 19 Fig. 1 Patch clamp recordings of Na + currents from hESC-derived RPE. a,bBrightfield light microscopy images of hESC-derived RPE cells. a Mature hESC-derived RPE grown on insert for 2 months showing strongly pigmented cells and characteristic epithelial morphology. bMature hESC-derived RPE was dissociated yielding single cells with typical morphology showing pigmented apical and non-pigmented basal sides. Scale bars 10 μm. Whole-cell patch clamp recordings as responses to a series of depolarizing voltage pulses (−80 to + 60 mV, 10 mV steps) after strong hyperpolarization (−170 mV) either cfrom mature monolayer of hESC-derived RPE or dfrom single hESC-derived RPE cells. Patch clamp pipette is visible in the center of the aand bimages. e–iAnalysis of the monolayer recordings. eThe average current–voltage relationship (Ivs V m , mean ± SEM, n= 12). fSteady-state inactivation curve was analyzed by plotting the normalized peak current at −10 mV test pulse against the prepulse voltage (−140 to −40 mV, 10 mV steps) and fitting the data with the Boltzmann equation. The best fit was obtained with V 1/2 =−94 ± 1 mV and k=10(n= 7). Data points indicate mean ± SEM. g, h The time dependency of recovery from inactivation. The second peak currents were normalized and plotted against the voltage pulse interval (10–270 ms). The best fit to an exponential function was obtained with τ=54±3ms (n= 5) (individual datapoints for havailable in Additional file 7: Table S2). iThe activation (squares) and inactivation (circles) time constants were obtained from single exponential fits to the rising and decaying phases of the current responses shown in cand plotted against the command voltage (n= 7). jSummary of the patch clamp results Johansson et al. BMC Biology (2019) 17:63 Page 3 of 19 Voltage-gated sodium channels localize near cell–cell junctions in RPE Our patch clamp data indicated that functional Na v channels are present in the hESC-derived RPE. The cellular localization of the channels was investigated by performing immunofluorescence studies where the cellular retinaldehyde-binding protein (CRALBP), a marker for RPE cells [16,17], was labeled together with the universal Na v channel marker. These hESC-derived RPE samples were then imaged with a laser scanning confocal Fig. 2 Blocker sensitivity and distribution of Na v channels. Patch clamp recordings were performed on mature hESC-derived RPE monolayers. aApplying TTX extracellularly (either 1 μMor10μM) did not entirely block the current (left). The current was completely removed by intracellular QX-314 (2 mM) (right). Laser scanning confocal microscopy (LSCM) images on Na v distribution in RPE cells. LSCM data inverted greyscale Z-maximum intensity projections of bhESC-derived and cmouse RPE stained against Na v channels (green) and RPE marker CRALBP (red). Scale bars 10 μm. dImmunogold labeling and transmission electron microscopy images showing Na v distribution at the apical membrane in the vicinity of the cell-cell junctions (black arrows). Scale bars 250 nm. eDissociated hESC-derived RPE cells were let to adhere to poly-l-lysine coated coverslips for 30 min, fixed and immunolabeled against Na v together with CRALBP (up) or tight junction marker ZO-1(down).TheNa v label concentrated on the belt-like region in the middle of the cell, between the basal and apical sides. Scale bars 5 μm Johansson et al. BMC Biology (2019) 17:63 Page 4 of 19 microscope (LSCM) by acquiring 3D image stacks (Fig. 2b), and the data were denoised by deconvolution. This showed that Na v channels were present in fully differentiated RPE. Furthermore, the Na v label concentrated primarily on the cellular borders with low expression elsewhere on the cell membrane while the CRALBP label was more uniformly localized to the apical side of the hESC-derived RPE (Fig. 2b). Since the expression of Na v channels in RPE has previously been thought to be induced in vitro by the cell culturing [18,19] and since cells derived from ESCs might not fully replicate the pattern of ion channel expression in vivo [11,20,24–28], we wanted to confirm their presence by using freshly isolated and non-cultured mouse RPE (Fig. 2c). The same labeling showed highly similar distributions in mouse RPE as in hESC-derived RPE: the CRALBP label was cytoplasmic on the apical side of the cells while Na v concentrated more on the cellular borders. Furthermore, the immunogold labeling for electron microscopy (immuno-EM) demonstrated the presence of Na v channels in the cell–cell junctions (Fig. 2d) and our immunolabeling with the tight junction marker ZO-1 showed highly overlapping distributions, strongly suggesting the primary Na v localization near the tight junctions (Additional file 1: Figure S1). We investigated the mechanism underlying the previously reported lack of Na v currents from acutely isolated RPE cells (Fig. 1d). The hESC-derived RPE cells were seeded on glass coverslips for 30 min and immunolabeled with the universal Na v marker, CRALPB and ZO1. Surprisingly, the Na v label was primarily concentrated in the narrow region separating the apical and basolateral sides of the cell. Together with ZO-1, Na v channels formed a clear ring-like structure between the apical and basal membranes following relaxation of junctional tension (Fig. 2e). Due to this junctional disruption, Na v channels might not be accessible to pass ionic currents in acutely dissociated RPE cells. RPE cells express various voltage-gated sodium channel subtypes Since ten different Na v channel subtypes, Na v 1.1–Na v 1.9 and Na x , have been identified with drastically different expression profiles in diverse cell types, we wanted to investigate which specific channel subtypes are functionally expressed in the RPE cells. At the mRNA level, previous work has detected all of the Na v channels in donated human RPE-choroid preparations, specifically Na v subtypes 1.2–1.6 and Na v 1.9 [29,30]. We performed immunolabeling experiments with mouse and hESCderived RPE using specific antibodies against channel subtypes Na v 1.1–Na v 1.9 (Fig. 3a, b, Additional file 2: Figure S2). Confocal microscopy showed that Na v 1.4 localizes as beads-on-a-string to the cell–cell junctions (Fig. 3a, b). Na v 1.8, on the other hand, localized overall to the apical side of the RPE cells (Fig. 3a, b). These data suggested that especially the Na v 1.4 and Na v 1.8 channels, which are usually expressed in skeletal muscle and dorsal root ganglia [31,32], respectively, are also present in RPE cells. Na v 1.6 the predominant channel of the adult central nervous system [33] showed a more homogenous labeling pattern in hESC-derived RPE and foci-like pattern in mouse RPE (Fig. 3a, b). Subtypes Na v 1.1, Na v 1.3, Na v 1.5, Na v 1.7, and Na v 1.9 were detected in cell–cell junctions and apical membrane but their labeling was more prominent after fixation with lower concentration of paraformaldehyde (Additional file 2: Figure S2). The subtype Na v 1.2 was only weakly detected in both hESC-derived and mouse RPE. Additionally, we investigated the changes in channel subtype localization patterns during maturation of hESC-derived RPE (Additional file 3: Figure S3). The immunolabeling experiments indicated that the subtypes Na v 1.4, Na v 1.5, and Na v 1.8 changed from homogeneous cellular distribution to more specific localization either to cell–cell junctions (Na v 1.4) or to the apical side of the epithelium (Na v 1.5 and Na v 1.8) during the first 9 days of maturation. To further verify the functional expression of the most prominent channel subtypes by electrophysiology, we repeated our patch clamp recordings using highly selective blockers for the channels Na v 1.4, Na v 1.6, and Na v 1.8. The average current–voltage relationship (I–Vcurve) was determined from all these recordings (n=7) (Fig. 3c). The current was sensitive to the combination of 30 nM 4,9-anhydro-TTX (Na v 1.6 blocker), 1μM A-803467 (Na v 1.8 blocker), and 600 nM μ-conotoxin GIIB (Na v 1.4 blocker), and the effect of inhibition was more potent with each added blocker thus confirming the expression and functionality of these channel subtypes in the hESC-derived RPE. However, the effect of inhibition was more significant when the blockers were combined with 10 μMTTXindicating the presence of Na v subtypes additional to 1.4, 1.6, and 1.8 (n= 11) (Fig. 3d). Finally, the channel subtype composition was verified by carrying out mass spectrometry (MS) analysis of gel bands obtained from hESC-derived RPE protein lysates that had been tested to show the main Na v subtypes by Western blot (Additional file 4:Figure S4). Here, we followed the “two-peptide rule”[34], considering a hit positive if two or more specific peptides were identified. Intriguingly, all of the nine types, except subtype Na v 1.2, were identified. This analysis thus further confirmed the expression of the three major subtypes (Na v 1.4, Na v 1.6, Na v 1.8) in RPE and was also positive for the Na x channel expression (Fig. 3e). Johansson et al. BMC Biology (2019) 17:63 Page 5 of 19 Voltage-gated sodium channels Na v 1.4 and Na v 1.8 are involved in POS phagocytosis in RPE Our previous experiments showed that several Na v subtypes are present in both mouse and mature hESC-derived RPE. However, their physiological relevance remained unknown. Phagocytosis of POS is one of the major roles of RPE [3], and a plausible candidate function for the Na v channels, as it requires rapid activation and high synchronization [35]. We therefore next investigated the potential importance of Na v channels for POS phagocytosis. To study their role in the phagocytosis process, we performed immunolabeling experiments with mouse eyes that had been prepared at light onset near the diurnal peak of phagocytosis. The role of the channels in POS uptake was studied by comparing the immunolabeling of the three major subtypes (Na v 1.4, Na v 1.6 and Na v 1.8) and opsin. Interestingly, at light onset, Na v 1.4 and Na v 1.8 localized to the bound POS particles (Fig. 4a). To confirm this redistribution of Na v channels, we next performed immuno-EM experiments (Fig. 4b, c), where we labeled the subtypes with gold nanoparticles in hESC-derived RPE. When the cells had not been exposed to POS particles, the localization of both channel subtypes was junction adjacent. This labeling pattern was particularly evident for Na v 1.4 (Fig. 4b) that formed clusters at the apical part of the cell–celljunctions.After2hor4hofphagocytosis, however, we could again observe the change in labeling distribution as the channels interacted directly with the phagocytic cups or recently ingested phagosomes (Fig. 4b, c). The redistribution of Na v channels occurring during phagocytosis (Fig. 5a) was studied ex vivo with the channel blockers (Fig. 5b). For this purpose, we developed an assay where freshly opened mouse eyecups were incubated in physiological conditions with blocker solutions for 1 h starting at 15 min prior to light onset. The blocker for Fig. 3 Immunolabeling of different Na v subtypes in hESC-derived and mouse RPE, mass-spectrometry studies of Na v expression, and patch clamp recordings with selective Na v blockers. a,bThe specific pattern of Na v subtypes was studied by immunolabeling. Laser scanning confocal microscopy Z-maximum intensity projections (xy-MIP) and yz cross-sections of amature hESC-derived or bmouse RPE. Na v subtypes 1.4, 1.6, and 1.8 (green) were immunolabeled together with filamentous actin (phalloidin stain, red). Scale bars 10 μm. Right side panels show a higher magnification of the highlighted regions. Patch clamp recordings were performed on mature hESC-derived RPE using selective blockers for channel subtypes. cNa v subtypes were sequentially blocked by extracellularly applied 4,9-AnhydroTTX (30 nM, Na v 1.6 blocker), A-803467 (1 μM, Na v 1.8 blocker) and μ-Conotoxin GIIB (600 nM, Na v 1.4 blocker). The average normalized peak current–voltage relationship (I/I max vs V m ) was determined from all recordings (mean ± SEM, n= 7). dApplying the selective blockers in combination with TTX (10 μM) removed most of the Na v currents (n= 11). eMass spectrometry analysis of Na v channel expression in hESC-derived RPE. Specific peptides were identified for all Na v subtypes, excluding Na v 1.2 Johansson et al. BMC Biology (2019) 17:63 Page 6 of 19 Na v 1.4 as well as the combination of all Na v blockers significantly prevented the disappearance of Na v 1.4 from cell– cell junctions when compared to the control (Fig. 5b). The inhibition effect was similarly observed in hESC-derived RPE in vitro when the cells were incubated for 2 h with POS mixed with blocker solutions (Fig. 5c). We did not observe significant differences in the overall labeling pattern of Na v 1.8 after the blocker incubation. Taken together, these experiments indicate the participation of Na v channels in the phagocytic processes of RPE cells in vitro and in vivo. Na v 1.4 knockdown and the inhibition of Na v channels significantly reduces the number of ingested POS particles in hESC-derived RPE Our LSCM and immuno-EM imaging of POS phagocytosis in RPE indicated a close interaction between Na v channels and phagocytosed POS particles. Therefore, we hypothesized that reducing the Na v channel activity could affect the rate of phagocytosis. After observing the dramatic change in the localization of Na v 1.4, we decided to study its effect further by silencing the channel subtype expression by shRNAs (Fig. 6). Due to the challenges associated with passaging of hESC-derived RPE cells, such as loss of the cobblestone morphology and poor cell proliferation, we opted for the lentivirus shRNA constructs. The transduction of the RPE cells had to be conducted several days after the cell seeding yielding a monolayer with sparse distribution of single GFP-positive cells (Fig. 6e). Since it was not possible to confirm the knockdown efficiency in hESC-derived RPE, the constructs were first validated with ARPE-19 cells (Additional file 5: Figure S5). The cells were transduced Fig. 4 POS phagocytosis and the role of Na v 1.4 and Na v 1.8. aPhagocytosis was studied by dissecting mouse eyes at various time points during the circadian cycle. Filamentous actin was stained with phalloidin (gray in the merged image) to highlight epithelial cell–cell junctions. Laser scanning confocal microscopy Z-maximum intensity projections of mouse RPE prepared at light onset showed localization of opsin labeled POS particles (blue) and Na v 1.4 (green) together with Na v 1.8 (red). Lower panels show a high contrast blowup of highlighted regions. Scale bars 10 μm. To study phagocytosis in vitro, mature hESC-derived RPE were labeled with 1.4 nm nanogold-conjugated antibodies against bNa v 1.4 and cNa v 1.8 during phagocytosis of purified porcine POS particles and in control conditions. Without POS exposure, both channels showed localization near the cell-cell junctions (black arrows) but by incubating the monolayers with POS particles for b2horc4 h, the localization (black arrows) was also evident around the phagocytic cups and recently ingested phagosomes. Scale bars 250 nm Johansson et al. BMC Biology (2019) 17:63 Page 7 of 19 with the shRNA constructs and collected for Western blot. Next, the knockdown effect of the verified construct was confirmed in hESC-derived RPE by conducting single cell patch clamp recordings and applying μ-conotoxin GIIB extracellularly (Fig. 6a-d). The cells expressing the target shRNA had both highly reduced Na v currents and minimal reactivity to the blocker (Fig. 6d) when compared to EGFPexpressing (Fig. 6c) or wildtype hESC-derived RPE cells (Fig. 6b). Intriguingly, when the cells were used in the phagocytosis assay (Fig. 6e), the silencing of Na v 1.4 caused a drastic reduction in the total number of POS particles found in individual GFP-positive cells on the monolayer (Fig. 6f, g). Fig. 5 Redistribution of Na v 1.4 during POS phagocytosis.The redistribution of Na v 1.4 during phagocytosis and the effect of Na v blockers to the process was studied in mouse and hESC-derived RPE. Filamentous actin was stained with phalloidin (red) to highlight epithelial cell-cell junctions. Laser scanning confocal microscopy Z-maximum intensity projections of aNa v 1.4 localization in mouse RPE at light onset and 2 h after it showed strong reduction of the beads-on-a-string type labeling from cell–cell junctions. Different assays were used to investigate Na v 1.4 distribution during phagocytosis and the effect of selective blockers for Na v 1.4 (600 nM μ-Conotoxin GIIB) and Na v 1.8 (1 μM A-803467) in combination with 10 μM TTX, or only of the selective blocker for Na v 1.4. bThe redistribution of Na v 1.4 was studied ex vivo by incubating opened mouse eyecups in control solution or with the selective blockers. In both of the blocker samples, the redistribution was inhibited and the beads-on-a-string type labeling remained visible (white arrows) in the cell-cell junctions. cThe hESC-derived RPE phagocytosis assay in vitro showed a highly similar redistribution of Na v 1.4 and the blockers had the same effect as in the ex vivo mouse eyecup assay. Scale bars 10 μm Johansson et al. BMC Biology (2019) 17:63 Page 8 of 19 To then study the effect of all Na v channels on a larger population of cells, we performed the in vitro phagocytosis assay (Fig. 7a) in the presence of Na v 1.4 and 1.8 blockers and TTX. The effect was first quantified by counting the number of particles from the immuno-EM images that had been tagged with gold nanoparticle labeled opsin (Fig. 7b). This revealed a drastic reduction in the total number of bound and internalized POS particles. To better analyze the effect, the assay was carried out by imaging large fields of immunolabeled opsin and ZO-1 and by comparing the number of POS particles in Na v blocker and control conditions after 2 h at +37 °C (Fig. 7c). The results showed that the blocker combination caused a 34% (n=18)reductioninthetotalnumberofPOS particles labeled with opsin (Fig. 7d). Fig. 6 POS phagocytosis assay of shRNA Na v 1.4 silenced hESC-derived RPE.Whole-cell patch clamp recordings were performed on mature hESCderived RPE monolayers as responses to a series of depolarizing voltage pulses (−80 to + 60 mV) after strong hyperpolarization afrom control RPE cells, control vector cells (EGFP) and cells where Na v 1.4 had been silenced with lentiviral vectors encoding shRNAs. The average current– voltage relationship (mean ± SEM,) was plotted for bControl hESC-derived RPE (n= 4), cEGFP expressing cells (n= 3), and dshRNA expressing cells (n= 3) (individual datapoints for b-davailable in Additional file 8: Table S3). eThe level of POS phagocytosis was analyzed with the EGFP expressing hESC-derived RPE cells. Filamentous actin was stained with phalloidin (blue) to highlight epithelial cell-cell junctions, EGFP (red) was used to identify the transduced cells and POS were labeled with opsin (green). fThe average distribution of POS particles was analyzed from several images that had a single shRNA expressing cell placed in the middle. gThe relative intensity of POS labeling in each square of the 3 × 3 grid was analyzed from Na v 1.4 shRNA cells (n= 22 images) and control EGFP cells (n= 18 images). Scale bars 10 μm Johansson et al. BMC Biology (2019) 17:63 Page 9 of 19 abundance threshold of more than 120 counts. Former target ions were excluded for 12 S.IDArolling collision energy (CE) parameters script was used for automatically controlling CE. Phagocytosis assay for hESC-derived and mouse RPE The porcine POS particles were isolated and purified as previously described [75,78]. Briefly, the eyecups obtained from a slaughterhouse were opened and retinas were removed using forceps under dim red light. The retinas were shaken gently in 0.73 M sucrose phosphate buffer and separated after filtering in sucrose gradient using an ultracentrifuge (Optima ultracentrifuge, Beckman Coulter, Inc., Brea, CA) at 112,400 x g for 1 h at +4 °C. The collected POS layer was centrifuged 3000×g for 10 min at +4 °C and stored in 73 mM sucrose phosphate buffer at −80 °C. The purified POS particles were fed to the hESCderived RPE cells in a KO-DMEM medium supplemented with 10% fetal bovine serum (FBS) and incubated for either 2 h at RT or 2 h, 4 h or 5 h at +37 °C in 5% CO 2 . In the blocker experiments, selective blockers for Na v 1.4, Na v 1.8 and TTX were also added to the medium for the incubation. Then the monolayers were washed twice briefly with PBS and fixed with PFA according to the immunostaining protocol. Phagocytosis was studied in vivo by preparing the mouse eyes under dim red light either at light onset or 2 h and 10 h after it. The mice were reared in normal 12-h light/dark cycle. When blockers were used, the eyecup was opened and then incubated in blocker solutions diluted in Ames’as described above, for 1 h at +37 °C with the retina left intact. Quantification of POS particles in hESC-derived RPE To detect and quantify POS particles, large random fields were imaged from 3 different samples in each condition with Zeiss LSM780 LSCM (the total number of images in each case is included in the figure legends as “n”). The images were first blurred with a Gaussian function after which a Z-maximum intensity projection was binarized using a global threshold. The number of POS particles was then analyzed from the images converted to mask. In the hESC-derived RPE where subtype Na v 1.4 had been silenced with lentiviral vectors encoding shRNAs, phagocytosis was analyzed by capturing several fields with a GFP-positive cell in the center of the image. The MIP images were then combined and the average distribution of POS particle labeling was compared between control-GFP (EGFP) construct and the clone TRCN000044419. The image was split into a 3 × 3 grid and the relative intensity of POS labeling was analyzed for each individual square of the grid. Statistical analysis of the POS phagocytosis quantification Each phagocytosis experiment was repeated three times and the images were pooled together. The normality of the data was tested by using Shapiro–Wilk normality test and the differences were first analyzed using ANOVA. Finally, pairwise comparison was conducted by using Kruskal–Wallis test to confirm the possible statistical significance between the experimental conditions. Confocal microscopy and image processing Confocal microscopy was performed with Zeiss LSM780 LSCM on inverted Zeiss Cell Observer microscope (Zeiss, Jena, Germany) by using Plan-Apochromat 63x/ 1.4 oil immersion objective. Voxel size was set to x=y= 66 nm and z= 200 nm and 1024 × 1024 pixel stacks of 70–120 slices were acquired with line average of 2. The Alexa Fluor 405 was excited with 405 nm diode laser; Alexa Fluor 488 with 488 nm laserline from Argon laser; Alexa Fluor 568 and TRITC with 561 nm DPSS or 562 nm InTune laser; Atto 633 and Alexa Fluor 647 with 633 nm HeNe and with 628 nm InTune laser. Emission was detected with windows of (in nm) 410–495 (DAPI, Alexa Fluor 405), 499–579 (Alexa Fluor 488), 579–642 (Alexa Fluor 568), and 642–755 (Alexa Fluor 647). Laser powers were minimized to avoid bleaching and photomultiplier tube sensitivities were adjusted to obtain optimal signal-to-noise ratio of the signal. The data was saved in .czi format and deconvolved using Huygens Essential (SVI, Hilversum, Netherlands) software. The deconvolution was performed with theoretical PSF, signalto-noise ratio of 5 and quality threshold of 0.01. Information regarding the refractive index of the sample was provided by the manufacturer of the ProLong Gold antifade mounting media. Images were further processed with ImageJ [79] and only linear brightness and contrast adjustments were performed for the pixel intensities. Final figures were assembled using Adobe Photoshop CC (2015.5.1 release) and Illustrator CC (2015.3.1 release) (Adobe Systems, San Jose, USA). Additional files Additional file 1: Figure S1. Immunolabeling of Na v in hESC-derived and mouse RPE. Z-maximum intensity projections (Z-MIP) of (a) hESCderived and (b) mouse RPE stained against Na v channels (green) and tight junction marker ZO-1 (red), together with cross-sectional X-MIPs from the highlighted regions. (PNG 729 kb) Additional file 2: Figure S2. Immunolabeling of different Na v subtypes in hESC-derived and mouse RPE. Different Na v channel subtypes were immunolabeled in (a) mature hESC-derived and (b) mouse RPE that had been fixed with 1% PFA. Laser scanning confocal microscopy Zmaximum intensity projections of Na v subtypes (green) labeled together with filamentous actin (phalloidin, red). In both samples, the subtypes Na v 1.1, Na v 1.3, Na v 1.5, Na v 1.7 and Na v 1.9 showed labeling in cell-cell junctions and apical membrane. The subtype Na v 1.2 gave extremely weak signals in both samples. Scale bars 10 μm. (PNG 4721 kb) Johansson et al. BMC Biology (2019) 17:63 Page 16 of 19 Additional file 3: Figure S3. Immunolabeling of different Na v subtypes during development of hESC-derived RPE. hESC-derived RPE cells were seeded on cell culture inserts and fixed at various timepoints during development. Laser scanning confocal microscopy Z-maximum intensity projections showed that during maturation from 1 d to 9 d after cell seeding, the cellular distribution of subtype Na v 1.1 stayed homogenous. Contrarily, cellular distribution of subtypes Na v 1.4 and Na v 1.5 changed from homogeneous (1 d) to more organized beads (9 d) at the cell-cell junctions (Na v 1.4) or to bright spots in the cell (Na v 1.5). The cellular distribution of Na v 1.8 was initially homogenous but at 9 d, the subtype also showed localization to one or few bright spots in the cells. Scale bars 10 μm. (PNG 1453 kb) Additional file 4: Figure S4. Western blot analysis of different subtypes in hESC-derived RPE. Whole cell lysates of hESC-derived RPE cells were analyzed by electroblotting and the resulting nitrocellulose membranes were stained against the subunits Na v 1.4-Na v 1.6 and Na v 1.8. All subunits showed positive bands between 130 and 250 kDa. The Western blots were used as guides for the gel excision for mass spectrometry analysis. (PNG 83 kb) Additional file 5: Figure S5. Western blot analysis of shRNA knockdown of Na V 1.4 in ARPE-19 cells. Whole cell lysates of ARPE-19 cells transduced with shRNA expressing EGFP or the lentivirus constructs were analyzed by Western blot. The nitrocellulose membranes were stained against the subunit Na v 1.4. The staining showed positive bands between 130 and 250 kDa for lysates obtained from EGFP expressing cells as well as cells transduced with shRNA clone 1 (TRCN0000416043) but the labeling intensity was decreased for lysates obtained from cells transduced with the clone 2 (TRCN0000425151) and especially with clone 3 (TRCN0000044419). The labeling band intensity was compared against the β-actin band (between 35 and 55 kDa) that was used as the loading control. Based on the Western blot, the expression for Na v 1.4 was normalized for EGFP and all shRNA constructs, and we therefore selected clone 3 (TRCN0000044419) for further experiments (Individual datapoints available in Additional file 9: Table S4). (PNG 328 kb) Additional file 6: Table S1. List of chemical and antibody details. (DOCX 46 kb) Additional file 7: Table S2. Individual datapoints for Fig. 1h. (DOCX 55 kb) Additional file 8: Table S3. Individual datapoints for Fig. 6b-d. (DOCX 69 kb) Additional file 9: Table S4. Individual datapoints for Figure S5. (DOCX 37 kb) Acknowledgements We would like to acknowledge the following contributors. We are grateful to Dr. Jari Hyttinen (Tampere University) for resources and support. We thank Drs. Kristian Donner (University of Helsinki) and Joshua Singer (University of Maryland) for valuable comments on the manuscript. We acknowledge Outi Heikkilä, Outi Melin, Hanna Pekkanen (all from Tampere University), Elina Hurskainen and Salla Mattola (both from University of Jyväskylä) for technical assistance and Dr. Hannele Uusitalo-Järvinen (Tampere University) as well as Dr. Petri Ala-Laurila's Lab (University of Helsinki) for providing the animal tissue. Tampere Facility of Electrophysiological Measurements, Tampere Imaging Facility, Tampere Mass Spectrometry Facility (Tampere University), and Electron Microscopy Unit (University of Helsinki, Institute of Biotechnology) are gratefully acknowledged for their services. Authors’contributions Conception and design of the study as well as data acquisition, analysis, and interpretation were performed by JKJ, VIJ, SH, AJ, TOI, and SN. The expertise on human embryonic stem cells and RPE differentiation was provided by HS, and the expertise on electron microscopy by SH and MV-R. The mass spectrometry expertise was provided by AJ and HU. All authors contributed to the writing of the manuscript with JKJ, TOI, and SN who are mainly responsible. All authors read and approved the final manuscript. Funding This work was supported by the Academy of Finland Grants 287287 (SN), 294054 (SN), 319257 (SN), 267471 (TOI), by Emil Aaltonen Foundation (SN), by Päivikki and Sakari Sohlberg Foundation (HS), and by Jane and Aatos Erkko Foundation (MV-R). Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Patch clamp, confocal imaging, and mass spectrometry datasets are available in the Zenodo repository [80]. Where n< 6, the individual data values are provided in additional files and cited in the figure legends (Additional file 7: Table S2, Additional file 8: Table S3, Additional file 9: Table S4). Ethics approval and consent to participate The National Authority for Medicolegal Affairs Finland has approved the study with human embryos (Dnro 1426/32/300/05). The supportive statement from the ethics committee of the Pirkanmaa hospital district Finland allows us to derive and expand hESC-lines from surplus embryos excluded from infertility treatments, and to use the lines for research purposes (R05116). New cell lines were not derived in this study. In mouse studies, all animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research using protocols approved and monitored by the Animal Experiment Board of Finland. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland. 2 Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland. 3 Tays Eye Centre, Tampere University Hospital, Tampere, Finland. Received: 25 March 2019 Accepted: 11 July 2019 References 1. Bok D. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci Suppl. 1993;17:189–95 http://www.ncbi.nlm.nih.gov/pubmed/8144697. Accessed 29 Aug 2016. 2. Steinberg RH. Interactions between the retinal pigment epithelium and the neural retina. Doc Ophthalmol. 1985;60:327–46 http://www.ncbi.nlm.nih. gov/pubmed/3905312. Accessed 29 Aug 2016. 3. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005;85:845–81. https://doi.org/10.1152/physrev.00021.2004. 4. Takei Y, Ozanics V. Origin and development of Bruch’s membrane in monkey fetuses: an electron microscopic study. Investig Ophthalmol. 1975; 14:903–16 http://www.ncbi.nlm.nih.gov/pubmed/811582. Accessed 1 Sep 2016. 5. Wimmers S, Karl MO, Strauss O. Ion channels in the RPE. Prog Retin Eye Res. 2007;26:263–301. https://doi.org/10.1016/j.preteyeres.2006.12.002. 6. Young RW, Bok D. Participation of the retinal pigment epithelium in the rod outer segment renewal process. JCell Biol. 1969;42:392–403. 7. Young RW. The daily rhythm of shedding and degradation of rod and cone outer segment membranes in the chick retina. Invest Ophthalmol Vis Sci. 1978;17:105–16 http://www.ncbi.nlm.nih.gov/pubmed/624604. Accessed 19 Feb 2017. 8. LaVail MM. Circadian nature of rod outer segment disc shedding in the rat. Invest Ophthalmol Vis Sci. 1980;19:407–11. http://www.ncbi.nlm.nih.gov/ pubmed/7358492. Accessed 19 Feb 2017. 9. Kevany BM, Palczewski K. Phagocytosis of retinal rod and cone photoreceptors. Physiology (Bethesda). 2010;25(1):8–15. https://doi.org/1 0.1152/physiol.00038.2009. 10. Müller C, Gómez NM, Ruth P, Strauß O. Ca V1.3 L-type channels, maxiK Ca (2+)-dependent K(+) channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells. Cell Signal. 2014;26:968–78. 11. Korkka I, Viheriälä T, Juuti-Uusitalo K, Uusitalo-Järvinen H, Skottman H, Hyttinen J, et al. Functional voltage-gated calcium channels are present in Johansson et al. BMC Biology (2019) 17:63 Page 17 of 19 human embryonic stem cell-derived retinal pigment epithelium. Stem Cells Transl Med. 2019;8(2):179–93. https://doi.org/10.1002/sctm.18-0026. 12. Karl MO, Kroeger W, Wimmers S, Milenkovic VM, Valtink M, Engelmann K, et al. Endogenous Gas6 and Ca2+-channel activation modulate phagocytosis by retinal pigment epithelium. Cell Signal. 2008;20:1159–68. https://doi.org/1 0.1016/J.CELLSIG.2008.02.005. 13. Fox JA, Pfeffer BA, Fain GL. Single-channel recordings from cultured human retinal pigment epithelial cells. J Gen Physiol. 1988;91:193–222 https://www. ncbi.nlm.nih.gov/pmc/articles/PMC2216133/. Accessed 23 Jan 2016. 14. Kokkinaki M, Sahibzada N, Golestaneh N. Human induced pluripotent stemderived retinal pigment epithelium (RPE) cells exhibition transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression pattern similar to native RPE. Stem Cells. 2011;29:825– 35. https://doi.org/10.1002/stem.635. 15. Sakai H, Saito T. Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: comparison with neuronal types of ion channels. J Neurobiol. 1997;32:377–90 http://www.ncbi.nlm.nih.gov/pubmed/9087890. Accessed 19 Feb 2017. 16. Bunt-Milam AH, Saari JC. Immunocytochemical localization of two retinoidbinding proteins in vertebrate retina. J Cell Biol. 1983;97:703–12 http://www. ncbi.nlm.nih.gov/pubmed/6350319. Accessed 26 Aug 2016. 17. Crabb JW, Carlson A, Chen Y, Goldflam S, Intres R, West KA, et al. Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein. Prorein Sci. 1998;7(3):746–57. https://www. ncbi.nlm.nih.gov/pmc/articles/PMC2143945/. 18. Botchkin LM, Matthews G. Voltage-dependent sodium channels develop in rat retinal pigment epithelium cells in culture. Proc Natl Acad Sci U S A. 1994;91:4564–8https://www.ncbi.nlm.nih.gov/pmc/articles/PMC43826/. Accessed 23 Jan 2016. 19. Wen R, Lui GM, Steinberg RH. Expression of a tetrodotoxin-sensitive Na+ current in cultured human retinal pigment epithelial cells. J Physiol. 1994; 476:187–96. https://doi.org/10.1113/jphysiol.1994.sp020122. 20. Reichhart N, Strauß O. Ion channels and transporters of the retinal pigment epithelium. Exp Eye Res. 2014;126:27–37. 21. Miyagishima KJ, Wan Q, Corneo B, Sharma R, Lotfi MR, Boles NC, et al. In pursuit of authenticity: induced pluripotent stem cell-derived retinal pigment epithelium for clinical applications. Stem Cells Transl Med. 2016;5: 1562–74. https://doi.org/10.5966/sctm.2016-0037. 22. Carrithers LM, Hulseberg P, Sandor M, Carrithers MD. The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations. FEMS Immunol Med Microbiol. 2011;63:319–27. https://doi.org/10.1111/j.1574-695 X.2011.00853.x. 23. Black JA, Waxman SG. Noncanonical roles of voltage-gated sodium channels. Neuron. 2013;80:280–91. https://doi.org/10.1016/j.neuron.2013.09.012. 24. Sachinidis A, Fleischmann BK, Kolossov E, Wartenberg M, Sauer H, Hescheler J. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc Res. 2003;58:278–91. https://doi.org/10.1016/S0008-6363(03)00248-7. Accessed 5 Mar 2019. 25. Wang K, Terrenoire C, Sampson KJ, Iyer V, Osteen JD, Lu J, et al. Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry. J Physiol. 2011; 589(Pt 24):6093–104. https://doi.org/10.1113/jphysiol.2011.220863. 26. Synnergren J, Améen C, Jansson A, Sartipy P. Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue. Physiol Genomics. 2012;44:245–58. https://doi.org/10.1152/physiolgenomics.00118.2011. 27. Kang J, Chen X-L, Ji J, Lei Q, Rampe D. Ca 2+ channel activators reveal differential L-type Ca 2+ channel pharmacology between native and stem cell-derived cardiomyocytes. J Pharmacol Exp Ther. 2012;341:510–7. https:// doi.org/10.1124/jpet.112.192609. 28. Hescheler J, Fleischmann BK, Lentini S, Maltsev VA, Rohwedel J, Wobus AM, et al. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc Res. 1997;36:149–62. https://doi. org/10.1016/S0008-6363(97)00193-4. Accessed 5 Mar 2019. 29. Booij JC, ten Brink JB, Swagemakers SMA, Verkerk AJMH, Essing AHW, van der Spek PJ, et al. A new strategy to identify and annotate human RPEspecific gene expression. PLoS One. 2010;5:e9341. https://doi.org/10.1371/ journal.pone.0009341. 30. Whitmore SS, Wagner AH, DeLuca AP, Drack AV, Stone EM, Tucker BA, et al. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq. Exp Eye Res. 2014;129: 93–106. https://doi.org/10.1016/j.exer.2014.11.001. 31. Bao L. Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons. Mol Pain. 2015;11:61. https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC4590712/. 32. Ptáček LJ, George AL, Griggs RC, Tawil R, Kallen RG, Barchi RL, et al. Identification of a mutation in the gene causing hyperkalemic periodic paralysis. Cell. 1991;67:1021–7. https://doi.org/10.1016/0092-86 74(91)90374-8. 33. Goldin AL. Diversity of mammalian voltage-gated sodium channels. Ann N Y Acad Sci. 1999;868(1):38–50. https://doi.org/10.1111/j.1749-6632.1999.tb11272.x. 34. Carr S, Aebersold R, Baldwin M, Burlingame A, Clauser K, Nesvizhskii A, et al. The need for guidelines in publication of peptide and protein identification data. Mol Cell Proteomics. 2004;3:531–3. https://doi.org/10.1074/mcp.T4 00006-MCP200. 35. Mazzoni F, Safa H, Finnemann SC. Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture. Exp Eye Res. 2014;126:51–60. https://doi.org/10.1016/j.exer.2014.01.010. 36. Mayerson PL, Hall MO. Rat retinal pigment epithelial cells show specificity of phagocytosis in vitro. J Cell Biol. 1986;103:299–308 http://www.ncbi.nlm.nih. gov/pubmed/3522605. Accessed 15 Jun 2018. 37. Cummins TR, Black JA, Dib-Hajj SD, Waxman SG. Glial-derived neurotrophic factor upregulates expression of functional SNS and NaN sodium channels and their currents in axotomized dorsal root ganglion neurons. J Neurosci. 2000;20:8754–61 http://www.ncbi.nlm.nih.gov/pubmed/11102483. Accessed 26 Feb 2019. 38. Black JA, Langworthy K, Hinson AW, Dib-Hajj SD, Waxman SG. NGF has opposing effects on Na+ channel III and SNS gene expression in spinal sensory neurons. Neuroreport. 1997;8:2331–5http://www.ncbi.nlm.nih.gov/ pubmed/9243635. Accessed 26 Feb 2019. 39. Catterall WA, Goldin AL, Waxman SG, International Union of Pharmacology. International Union of Pharmacology. XXXIX. Compendium of voltage-gated ion channels: sodium channels. Pharmacol Rev. 2003;55:575–8. https://doi. org/10.1124/pr.55.4.7. 40. Noda M, Hiyama TY. The Na x channel. Neurosci. 2015;21:399–412. https:// doi.org/10.1177/1073858414541009. 41. Herzog RI, Cummins TR, Ghassemi F, Dib-Hajj SD, Waxman SG. Distinct repriming and closed-state inactivation kinetics of Nav1.6 and Nav1.7 sodium channels in mouse spinal sensory neurons. J Physiol. 2003;551:741– 50. https://doi.org/10.1113/jphysiol.2003.047357. 42. Cummins TR, Aglieco F, Renganathan M, Herzog RI, Dib-Hajj SD, Waxman SG. Nav1.3 sodium channels: rapid repriming and slow closed-state inactivation display quantitative differences after expression in a mammalian cell line and in spinal sensory neurons. J Neurosci. 2001;21: 5952–61. https://doi.org/10.1523/JNEUROSCI.21-16-05952.2001. 43. Ishida T, Takei R, Gautam SH, Otsuguro K, Ohta T, Ito S, et al. Voltage-gated channel properties of epithelial cells in porcine vomeronasal organ. Neurosci Lett. 2008;441:277–81. https://doi.org/10.1016/j.neulet.2008.06.045. 44. Sontheimer H, Black JA, Waxman SG. Voltage-gated Na+ channels in glia: properties and possible functions. Trends Neurosci. 1996;19:325–31 http:// www.ncbi.nlm.nih.gov/pubmed/8843601. Accessed 14 Mar 2019. 45. Sontheimer H, Waxman SG. Ion channels in spinal cord astrocytes in vitro. II. Biophysical and pharmacological analysis of two Na+ current types. J Neurophysiol. 1992;68:1001–11. https://doi.org/10.1152/jn.1992.68.4.1001. 46. Sontheimer H, Waxman SG. Expression of voltage-activated ion channels by astrocytes and oligodendrocytes in the hippocampal slice. 1993. https://doi. org/10.1152/jn.1993.70.5.1863. 47. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003; 422:198–207. https://doi.org/10.1038/nature01511. 48. Shevchenko A, Loboda A, Ens W, Schraven B, Standing KG, Shevchenko A. Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry. Electrophoresis. 2001;22:1194–203. https://doi.org/1 0.1002/1522-2683()22:6<1194::AID-ELPS1194>3.0.CO;2-A. 49. HavlišJ, Shevchenko A. Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry. Anal Chem. 2004;76:3029–36. https://doi.org/10.1021/ac035286f. 50. Wilkinson TCI, Gardener MJ, Williams WA. Discovery of functional antibodies targeting ion channels. J Biomol Screen. 2015;20:454–67. https://doi.org/1 0.1177/1087057114560698. 51. Konno K, Watanabe M. Immunohistochemistry for Ion Channels and Their Interacting Molecules: Tips for Improving Antibody Accessibility. Johansson et al. BMC Biology (2019) 17:63 Page 18 of 19 New York: Humana Press; 2016. p. 171–8. https://doi.org/10.1007/978-1-4 939-3064-7_13. 52. Craner MJ, Damarjian TG, Liu S, Hains BC, Lo AC, Black JA, et al. Sodium channels contribute to microglia/macrophage activation and function in EAE and MS. Glia. 2005;49:220–9. https://doi.org/10.1002/glia.20112. 53. Carrithers MD, Dib-Hajj S, Carrithers LM, Tokmoulina G, Pypaert M, Jonas EA, et al. Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification. J Immunol. 2007;178:7822–32 http://www.ncbi.nlm.nih.gov/pubmed/17548620. Accessed 11 Mar 2019. 54. Carrithers MD, Chatterjee G, Carrithers LM, Offoha R, Iheagwara U, Rahner C, et al. Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A. J Biol Chem. 2009;284:8114–26. https://doi. org/10.1074/jbc.M801892200. 55. Nandrot EF, Kim Y, Brodie SE, Huang X, Sheppard D, Finnemann SC. Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin. J Exp Med. 2004;200:1539–45. https://doi.org/10.1084/ jem.20041447. 56. Craner MJ, Lo AC, Black JA, Waxman SG. Abnormal sodium channel distribution in optic nerve axons in a model of inflammatory demyelination. Brain. 2003;126:1552–61. https://doi.org/10.1093/brain/awg153. 57. Black JA, Liu S, Waxman SG. Sodium channel activity modulates multiple functions in microglia. Glia. 2009;57:1072–81. https://doi.org/1 0.1002/glia.20830. 58. Karl M, Kroeger W, Wimmers S, Milenkovic V, Valtink M, Engelmann K, Strauss O. Endogenous Gas6 and Ca2+-channel activation modulate phagocytosis by retinal pigment epithelium. Cellular Signalling. 2008;20(6): 1159–68. https://doi.org/10.1016/J.CELLSIG.2008.02.005. 59. Gronski MA, Kinchen JM, Juncadella IJ, Franc NC, Ravichandran KS. An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response. Cell Death Differ. 2009;16:1323–31. https://doi.org/10.1038/cdd.2009.55. 60. Mangini NJ, Haugh-Scheidt L, Valle JE, Cragoe EJ, Ripps H, Kennedy BG. Sodium-calcium exchanger in cultured human retinal pigment epithelium. Exp Eye Res. 1997;65:821–34 http://www.ncbi.nlm.nih.gov/pubmed/9441706. Accessed 20 Jun 2018. 61. Fijisawa K, Ye J, Zadunaisky JA. A Na+/Ca2+ exchange mechanism in apical membrane vesicles of the retinal pigment epithelium. Curr Eye Res. 1993;12:261–70 http://www.ncbi.nlm.nih.gov/pubmed/8482115. Accessed 20 Jun 2018. 62. Araujo EG, Persechini PM, Oliveira-Castro GM. Electrophysiology of phagocytic membranes. Role of divalent cations in membrane hyperpolarizations of macrophage polykaryons. Biochim Biophys Acta Biomembr. 1986;856:362–72. https://doi.org/10.1016/0005-2736(86)90047-7. 63. Ince C, Coremans JM, Ypey DL, Leijh PC, Verveen AA, van Furth R. Phagocytosis by human macrophages is accompanied by changes in ionic channel currents. J Cell Biol. 1988;106:1873–8. https://doi.org/10.1083/JCB.1 06.6.1873. 64. Marshansky V, Futai M. The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function. Curr Opin Cell Biol. 2008;20:415–26. https://doi.org/10.1016/J.CEB.2008.03.015. 65. Steinberg BE, Touret N, Vargas-Caballero M, Grinstein S. In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force. Proc Natl Acad Sci. 2007; 104:9523–8. https://doi.org/10.1073/pnas.0700783104. 66. Xu H, Ren D. Lysosomal physiology. Annu Rev Physiol. 2015;77:57–80. https://doi.org/10.1146/annurev-physiol-021014-071649. 67. Catterall WA. Signaling complexes of voltage-gated sodium and calcium channels. Neurosci Lett. 2010;486:107–16. https://doi.org/10.1016/J.NEULET.2 010.08.085. 68. Abu Khamidakh AE, Juuti-Uusitalo K, Larsson K, Skottman H, Hyttinen J. Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation. Exp Eye Res. 2013;108:129–39. https://doi.org/10.1016/j.exer.2013.01.009. 69. Churchill GC, Atkinson MM, Louis CF. Mechanical stimulation initiates cell-tocell calcium signaling in ovine lens epithelial cells. J Cell Sci. 1996:355–65 http://www.ncbi.nlm.nih.gov/pubmed/8838659. Accessed 19 Feb 2017. 70. Stalmans P, Himpens B. A decreased Ca2+−wave propagation is found among cultured RPE cells from dystrophic RCS rats. Invest Ophthalmol Vis Sci. 1998;39:1493–502 http://www.ncbi.nlm.nih.gov/pubmed/9660499. Accessed 19 Feb 2017. 71. Wang C, Chung BC, Yan H, Wang H-G, Lee S-Y, Pitt GS. Structural analyses of Ca2+/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation. Nat Commun. 2014;5:4896. https://doi.org/1 0.1038/ncomms5896. 72. Adams PJ, Ben-Johny M, Dick IE, Inoue T, Yue DT. Apocalmodulin itself promotes ion channel opening and Ca2+ regulation. Cell. 2014;159:608–22. https://doi.org/10.1016/j.cell.2014.09.047. 73. Langer J, Stephan J, Theis M, Rose CR. Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ. Glia. 2012;60: 239–52. https://doi.org/10.1002/glia.21259. 74. Chao TI, Skachkov SN, Eberhardt W, Reichenbach A. Na + channels of Müller (glial) cells isolated from retinae of various mammalian species including man. Glia. 1994;10:173–85. https://doi.org/10.1002/glia.440100304. 75. Vaajasaari H, Ilmarinen T, Juuti-Uusitalo K, Rajala K, Onnela N, Narkilahti S, et al. Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells. Mol Vis. 2011; 17:558–75 http://www.ncbi.nlm.nih.gov/pubmed/21364903. Accessed 29 Aug 2016. 76. Pohl K, Stierhof Y-D. Action of gold chloride (“gold toning”) on silverenhanced 1 nm gold markers. Microsc Res Tech. 1998;42:59–65. https://doi. org/10.1002/(SICI)1097-0029(19980701)42:1<59::AID-JEMT7>3.0.CO;2-M. 77. Sawada H, Esaki M. A practical technique to postfix nanogoldimmunolabeled specimens with osmium and to embed them in Epon for electron microscopy. J Histochem Cytochem. 2000;48:493–8. https://doi. org/10.1177/002215540004800407. 78. Mao Y, Finnemann SC. Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture. Methods Mol Biol. 2013;935:285–95. https://doi.org/10.1007/978-1-62703-080-9_20. 79. Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5. https://doi.org/10.1038/nmeth.2089. 80. Johansson JK, Karema-Jokinen V, Hakanen S, Jylha A, Uusitalo H, VihinenRanta M, et al. Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium. 2019. https://doi.org/10.52 81/ZENODO.3270652.Accessed09July2019. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Johansson et al. BMC Biology (2019) 17:63 Page 19 of 19