Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium
Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav 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 Nav 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 Nav channels in RPE both ex vivo and in vitro.
Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. 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 Nav subtypes. Importantly, we show that the Nav 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 Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process.
Taken together, our study shows that Nav 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.
KeywordsRPE Ion channels Nav Patch clamp Phagocytosis Retina Photoreceptors
In the vertebrate eye, the retinal pigment epithelium (RPE) forms a barrier between the retina and the choroid [1, 2, 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  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 . Recent studies imply the importance of specific ion channels in this process including the L-type calcium channels as well as calcium-dependent potassium and chloride channels [10, 11, 12].
Since the first single-cell recordings from RPE in 1988 , a large variety of different ion channels have been identified in them . Among these are several voltage-gated calcium, potassium, and chloride channels. However, the identity of sodium conductive ion channels in RPE has remained elusive , 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+ (Nav) channels in RPE [5, 14, 15, 18, 19]. However, electrophysiological data demonstrating their functionality is missing in mature RPE. More importantly, Nav 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 the presence of Nav channels both in cultured human embryonic stem cell (hESC)-derived RPE and freshly isolated mouse RPE. We show that Nav channels co-regulate photoreceptor outer segment (POS) phagocytosis. Our hypothesis is supported by a recent demonstration of the involvement of Nav channels in phagocytosis of mycobacteria by macrophages . Our work provides evidence that Nav1.8 accumulates with the phagosomal particles. Nav1.4 also accumulates to phagosomes but displays localization to cell–cell junctions outside phagocytosis. Interestingly, selective Nav channel blockers significantly reduced this phagosomal translocation. Moreover, the selective blockers combined with the universal Nav 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 Nav1.4 was silenced with short hairpin RNA (shRNA). More generally, our observations add to the growing body of evidence that Nav 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 ). Collectively, our results show that this epithelium is electrically more complex than was previously thought.
Functional voltage-gated sodium channels are present in RPE derived from human embryonic stem cells
Voltage-gated sodium channels localize near cell–cell junctions in RPE
Our patch clamp data indicated that functional Nav 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 Nav channel marker. These hESC-derived RPE samples were then imaged with a laser scanning confocal microscope (LSCM) by acquiring 3D image stacks (Fig. 2b), and the data were denoised by deconvolution. This showed that Nav channels were present in fully differentiated RPE. Furthermore, the Nav 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 Nav 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, 25, 26, 27, 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 Nav concentrated more on the cellular borders. Furthermore, the immunogold labeling for electron microscopy (immuno-EM) demonstrated the presence of Nav 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 Nav localization near the tight junctions (Additional file 1: Figure S1).
We investigated the mechanism underlying the previously reported lack of Nav 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 Nav marker, CRALPB and ZO-1. Surprisingly, the Nav label was primarily concentrated in the narrow region separating the apical and basolateral sides of the cell. Together with ZO-1, Nav 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, Nav channels might not be accessible to pass ionic currents in acutely dissociated RPE cells.
RPE cells express various voltage-gated sodium channel subtypes
Subtypes Nav1.1, Nav1.3, Nav1.5, Nav1.7, and Nav1.9 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 Nav1.2 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 Nav1.4, Nav1.5, and Nav1.8 changed from homogeneous cellular distribution to more specific localization either to cell–cell junctions (Nav1.4) or to the apical side of the epithelium (Nav1.5 and Nav1.8) 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 Nav1.4, Nav1.6, and Nav1.8. The average current–voltage relationship (I–V curve) was determined from all these recordings (n = 7) (Fig. 3c). The current was sensitive to the combination of 30 nM 4,9-anhydro-TTX (Nav1.6 blocker), 1 μM A-803467 (Nav1.8 blocker), and 600 nM μ-conotoxin GIIB (Nav1.4 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 μM TTX indicating the presence of Nav 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 Nav subtypes by Western blot (Additional file 4: Figure S4). Here, we followed the “two-peptide rule” , considering a hit positive if two or more specific peptides were identified. Intriguingly, all of the nine types, except subtype Nav1.2, were identified. This analysis thus further confirmed the expression of the three major subtypes (Nav1.4, Nav1.6, Nav1.8) in RPE and was also positive for the Nax channel expression (Fig. 3e).
Voltage-gated sodium channels Nav1.4 and Nav1.8 are involved in POS phagocytosis in RPE
Our previous experiments showed that several Nav 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 , and a plausible candidate function for the Nav channels, as it requires rapid activation and high synchronization . We therefore next investigated the potential importance of Nav channels for POS phagocytosis.
Nav1.4 knockdown and the inhibition of Nav channels significantly reduces the number of ingested POS particles in hESC-derived RPE
Next, we wanted to independently quantify the binding and internalization stages of the phagocytosis pathway. The binding was investigated by incubating the POS-supplemented hESC-derived RPE cells at room temperature (RT) [35, 36] with 5% CO2. Most of the unbound POS were then removed by gentle washing, and the monolayers were placed in the incubator for either 2 h or 5 h to investigate the internalization stage of phagocytosis. This was carried out also with the combination of Nav blockers and the total number of POS particles was compared to control cells at each timepoint (Fig. 7e, f). The results showed no statistically significant changes (p = 0.1) in the number of POS particles due to the Nav inhibition in the particle binding phase (2 h RT, Control n = 15, Nav blockers n = 10). However, Nav blocking caused a 41% reduction of POS particle numbers in the internalization phase (2 h RT + 2 h at +37 °C, Control n = 15, Nav blocker n = 15) (p < 0.001). In control cells, the internalized particles were degraded, which was detected as a 25% reduction in particle numbers in the processing phase (2 h RT + 5 h at +37 °C, n = 15, p < 0.01) (Fig. 7f). Nav blocking substantially reduced further processing of POS particles in RPE cells as there were no significant changes in particle numbers between internalization and processing phases (n = 15, p = 0.16). Together, our data indicate that functional Nav channels directly interact with phagosomes (Fig. 4) and that they are involved in the POS particle internalization and further processing (Fig. 7e, f).
Recent studies show revolution in our understanding of the roles that Nav channels have in cellular functions; no longer are these proteins considered important only in “classically” electrically excitable tissues. Here, we provide the first evidence, to our knowledge, that Nav channels are expressed in the epithelial cells (Figs. 1, 2, and 3, Additional file 1: Figure S1, Additional file 2: Figure S2) and that their activity co-regulates phagocytosis in RPE. Our observations of Nav channels and Nav-mediated currents in intact RPE preparations (mature hESC-derived RPE monolayers and freshly isolated mouse RPE) demonstrate that previous observations of Nav-mediated currents in cultured RPE cells are not preparation-dependent artifacts [18, 19]. Rather, the absence of Nav-mediated currents in acutely isolated RPE cells (Fig. 1d) likely results from the destruction of tight junction complexes during dissociation (Fig. 2e). Internalization of Nav channels, of course, would result in diminution or absence of membrane currents mediated by these channels as observed by us (Fig. 1d) and others [18, 19]. Reductions in Nav currents have also been reported in dorsal root ganglion neurons following peripheral axotomy [37, 38] but this effect was found to be reversible by exposure to growth factors [37, 38]. The observation of Nav currents in recordings from hESC-derived RPE monolayers, we believe, is strong evidence that cells in RPE with intact tight junctions usually express functional Nav channels in their plasma membranes.
The properties of Nav-mediated currents in hESC-derived RPE cells are consistent with the earlier recordings from other non-neuronal cells . Furthermore, non-excitable cells have been shown to display varying sensitivity to TTX based on their Nav subtype composition , and our recordings from RPE align with this observation. The high TTX concentration needed for a full inhibition of the current is supported by the finding that RPE cells were labeled strongly by an anti-Nav1.8 antibody; Nav1.8 is the least sensitive of Nav channels to TTX . Our pharmacological analysis of Nav-mediated currents using Nav subtype-specific blockers, immunofluorescence microscopy and MS-based analysis (Fig. 3, Additional file 2: Figure S2) indicated that Nav1.1 as well as Nav1.3–Nav1.9 subtypes are present in the RPE together with the sodium concentration sensitive Nax channel . This data was further supported by the Nav current characteristics: the relatively slow recovery from inactivation [41, 42] (Fig. 1h) and the large variation visible in the early phase of the recovery, indicate the presence of several different Nav subtypes in the RPE. Compared to neurons, other non-excitable cells, such as non-process bearing astrocytes and supporting cells of the porcine vomeronasal organ [43, 44, 45], have been shown to exhibit more negative steady-state half-inactivation voltages, and similar V1/2 values were found for hESC-derived RPE (Fig. 1f). Accordingly, and similarly to astrocytes , we never observed spontaneous action potentials in these cells. Earlier recordings from RPE have shown V1/2 values much closer to neurons; however, these results were obtained after only a short period in culture [15, 19]. As our data demonstrates that the Nav channel localization and subtype composition is dynamically regulated during development (Additional file 3: Figure S3), we believe that RPE maturation stage including the junctional localization of the channels could greatly influence their electrophysiological properties. This is supported by the previous recordings that identified Nav channels in astrocytes in hippocampal slices .
For the MS-based identification of Nav proteins, we chose to perform the analysis from gel bands [47, 48, 49] due to the high sensitivity and possibility for absolute protein identification  provided by this approach. However, it was challenging to purify the Nav channel proteins from the RPE cells, most probably due to their several transmembrane domains and high hydrophobicity . Therefore, the number of detected Nav channel-specific peptides does not necessarily directly correlate to their relative abundance. Nav1.4 and Nav1.8 of the identified Nav subtypes showed the strongest staining in the immunohistochemical analysis and carried over one third of the total current in patch clamp recordings. Therefore, we focused on these two channel subtypes while investigating the physiological roles of Nav channels. It is noteworthy, however, that the subtypes Nav1.1, Nav1.3, Nav1.5, and Nav1.7 only showed strong junctional labeling after fixation with 1% PFA. Our antibodies labeling these subtypes are directed against different residues of the same intracellular loop between the domains III and IV. Importantly, this region might be sequestered in the dense cell–cell junctions thus hampering their detection through conventional immunocytochemistry . On the other hand, cells derived from ESCs can bear differences compared to native cells regarding subtype composition. Earlier studies have demonstrated high similarity but also certain deviations in terms of ion channel distribution and biophysical characteristics as well as channel pharmacology and transcriptional profile [11, 20, 24, 25, 26, 27, 28].
Previous studies have shown that in macrophages, Nav channels have important roles in phagocytosis [22, 52, 53, 54]. We observed an accumulation of Nav1.4 and Nav1.8 towards POS particles during phagocytosis. The translocation of Nav1.4 from tight junctions was still evident 2 h after light onset (Fig. 5a), near the peak expression level of a phagocyte cell surface tyrosine kinase receptor MerTK . The involvement of Nav1.4 in phagocytosis was supported by the fact that its silencing decreased the amount of POS particles (Fig. 6). Furthermore, following Nav blocker incubation, we observed a decrease in its translocation (Fig. 5) with a concurrent reduction in the number of POS particles (Fig. 7). Nav channel redistribution has been observed in demyelinated axonal membrane  suggesting that these ion channels can display dynamic regulation of distribution and have important implications in various pathologies.
Participation of Nav channel activity to POS phagocytosis was further indicated by their direct association with both the forming phagocytic cups and ingested phagosomes (Fig. 4). Although inhibition of Nav channels did not abolish phagocytosis, the observed 41% attenuation (Fig. 7) was similar to the previously reported effect of TTX in microglia . Our assays suggest that the Nav channels are involved in the engulfment and further processing of the phagosomes, since inhibiting Nav activity did not impair the binding of POS particles (Fig. 7). Interestingly, changes in intracellular free calcium concentration regulate phagocytosis in RPE  and, more specifically, particle engulfment in other phagocytes . Nav mediated sodium influx could result in increased calcium concentration via reversed functioning of sodium–calcium exchangers expressed in the RPE apical membrane [60, 61] thus affecting phagocytosis. Alternatively, Nav channels could regulate endosomal acidification by providing a sodium efflux pathway to enhance the entry of protons, similarly to macrophages [22, 53]. In these cells, phagocytosis has been associated with membrane potential hyperpolarization due to the activation of a Ca2+-dependent K+ conductance [62, 63]. Such change in potential could relieve the Nav channel inactivation in the early phase of phagocytosis, and the channels could subsequently be activated in the phagosomes and endosomes with membrane potential in the Nav activation range [64, 65, 66]. Furthermore, the channels could be involved directly in the circadian control of the pathway, as has been recently shown for other ion channels .
The fact that RPE expresses such a versatile array of Nav channels suggests that besides phagocytosis, these channels also have other roles in the physiology of the RPE. Overall, sodium homeostasis is critical to epithelial transport mechanisms, and our observation of Nav channels, including the non-voltage-gated Nax channel, brings a new piece in the ongoing identification of the sodium conducting proteins in RPE. In excitable cells, Nav and Ca2+ channels form local signaling complexes that are essential for various intracellular processes . Similar roles for these channels could be possible in RPE as well. Moreover, epithelial cells, including RPE, show strong calcium waves in response to mechanical stimulation [68, 69, 70], and it is likely that Nav channels are involved in the process. It is well established that the Ca2+ binding protein calmodulin (Cam) interacts directly with the C-terminal domain of Nav , and it was recently shown that the Ca2+-free form of Cam, ApoCam, enhances the Nav channel opening by several-fold . Thus, Na+- and Ca2+-dependent signaling pathways can interact in epithelia as has been reported in the case of astrocytes . Lastly, it has been suggested that retinal Müller cells that display similar low Nav channel densities could be activated by the adjacent neurons and it is possible that RPE cells could also serve as voltage sensors reacting to signals arising from photoreceptors [44, 74].
The results of this study demonstrate that functional Nav channels are present in mouse and hESC-derived RPE cells with intact tight junctions. Specifically, we confirm the presence of Nav1.1 as well as Nav1.3–Nav1.9 subtypes and the sodium concentration sensitive Nax channel, showing that their expression is not due to specific culturing conditions. Our data shows that the most prominent subtypes Nav1.4 and Nav1.8 are involved in photoreceptor outer segment renewal by directly interacting with phagosomes. Inhibiting the activity of these channels by either pharmacological blockers or shRNA-mediated silencing impairs the phagocytosis process, particularly at the engulfment or further processing stages. Collectively, we demonstrate that Nav channels yield RPE cells the capacity for fast voltage sensitivity and that the channels are a vital part of its physiology.
Antibodies and reagents
Catalog and batch numbers as well as other information for the chemicals and antibodies used in this study can be found from the Additional file 6: Table S1.
Human ESC lines Regea08/023 and Regea08/017 were cultured as previously described [11, 75]. Briefly, the hESC-derived RPE were spontaneously differentiated in floating cell clusters. The pigmented areas were isolated manually and the cells were dissociated with Tryple Select (1X, Thermo Fisher Scientific) and filtered through cell strainer (BD Biosciences, NJ, USA). The isolated cells were then seeded on collagen IV-coated (human placenta, 5 μg/cm2; Sigma-Aldrich, MO, USA) 24-well plates (NUNC, Thermo Fisher Scientific, Tokyo, Japan) for enrichment. Subsequently, the pigmented cells were replated for maturation on culture inserts (Millicell Hanging Cell Culture Insert, polyethylene terephthalate, 1.0 μm pore size, EMD Millipore, MA, USA) coated either with Collagen IV (10 μg/cm2) or with collagen IV and laminin (1.8 μg/cm2, LN521, Biolamina, Sweden). The cells were cultured at +37 °C in 5% CO2 in culture medium consisting of Knock-Out Dulbecco’s modified Eagle’s medium (KO-DMEM), 15% Knock-Out serum replacement (KO-SR), 2 mM GlutaMax, 0.1 mM 2-mercaptoethanol (all from Life Technologies, Carlsbad, CA), 1% Minimum Essential Medium nonessential amino acids, and 50 U/mL penicillin/streptomycin (from Cambrex BioScience, Walkersville, MD, USA). The culture medium was replenished three times a week. Mature monolayers typically showed transepithelial resistance values (TER) of over 200 Ω cm2.
Generation of Nav1.4 shRNA cultures
After Western blot analysis, 2 μl of the verified clone TRCN000044419 (8.1 × 106 TU/ml) and 8 mg/ml polybrene were added on hESC-derived RPE cells grown on insert. Transduction was made 5–23 days after the cell seeding and virus particles were incubated for 1 day before changing medium. The silencing of Nav1.4 current was verified by patch clamp from maturated, 8–10 weeks old, hESC-derived RPE cells.
For monolayer patch clamp recordings and immunolabeling, the membrane of the culture insert was removed from the insert holder and cut into smaller pieces. The cells were rinsed three times either with PBS (for immunolabeling) or with Ames’ solution (for patch clamp recordings). For the experiments on dissociated cells, the hESC-derived RPE monolayers were treated with TrypLE Select for 10 min in +37 °C, gently mechanically triturated with a pipette and centrifuged for 5 min at 1000 rpm. Dissociated cells were resuspended in culture medium, seeded on glass coverslips coated with poly-l-lysine (Sigma-Aldrich) and allowed to settle down for 10 min for patch clamp recordings and 30 min for immunolabeling.
Mouse RPE was prepared for immunolabeling as follows. C57BL/6 mice were euthanized by CO2 inhalation and cervical dislocation. The eyes were enucleated and bisected along the equator, and the eyecups were sectioned in Ames’ solution buffered with 10 mM HEPES and supplemented with 10 mM NaCl, pH was adjusted to 7.4 with NaOH (Sigma-Aldrich). The retina was gently removed from the eyecup leaving the RPE firmly attached to the eyecup preparation.
Patch clamp recordings
Ionic currents were recorded from mature hESC-derived RPE monolayers or freshly dissociated cells using the standard patch clamp technique in whole-cell configuration. Patch pipettes (resistance 5–6 MΩ) were filled with an internal solution containing (in mM) 83 CsCH3SO3, 25 CsCl, 10 TEA-Cl, 5.5 EGTA, 0.5 CaCl2, 4 ATP-Mg, 0.1 GTP-Na, 10 HEPES, and 5 NaCl; pH was adjusted to ~ 7.2 with CsOH and osmolarity was ~ 290 mOsm (Gonotec, Osmomat 030, Labo Line Oy, Helsinki, Finland). For the recordings with K+-based internal solution, CsCl was replaced with KCl and CsCH3SO3 was replaced with K-gluconate. In some experiments, the internal solution also contained 2 mM QX-314-Cl (from Sigma-Aldrich). During all recordings, the tissue was perfused at 2.5 ml min−1 with Ames’ solution (Sigma-Aldrich) buffered with 10 mM HEPES and supplemented with 10 mM NaCl and 5 mM TEA-Cl. The pH was adjusted to 7.4 with NaOH and the osmolarity set to ~ 305 mOsm. The bath solution contained 10 nM-10 μM TTX citrate (from Tocris Bioscience) when the effect of TTX on the recorded currents was investigated, and 30 μM 18α-glycyrrhetinic acid (from Sigma-Aldrich) when the effect of gap junctional coupling was tested. For the channel subtype recordings, the bath solution was supplemented with 30 nM 4,9-AnhydroTTX, 1 μM A-803467, or 600 nM μ-Conotoxin GIIB. All recordings were made in voltage clamp mode with pClamp 10.2 software using the Axopatch 200B patch clamp amplifier connected to an acquisition computer via AD/DA Digidata 1440 (Molecular Devices, USA). The access resistance was below 30 MΩ and the membrane resistance above 150 MΩ. Series resistance was 15–30 MΩ and was not compensated. Holding potentials were corrected for a 3 mV liquid junction potential during the data analysis. All recordings were performed at room temperature.
Prior to immunolabeling, samples were washed three times with PBS and fixed for 15 min with 4% paraformaldehyde or 10 min with 1% paraformaldehyde (pH 7.4; Sigma-Aldrich). After repeated washes with PBS, samples were permeabilized by incubating in 0.1% Triton X-100 in PBS (Sigma-Aldrich) for 15 min and subsequently blocked with 3% BSA (BSA; Sigma-Aldrich) for 1 h. All immunolabeling incubations were done at room temperature.
Primary antibodies against the following proteins were used in this study: cellular retinaldehyde-binding protein (CRALBP) 1:400 (ab15051, Abcam), Nav1.1 1:200 (ASC-001, Alomone labs), Nav1.2 1:200 (ab99044, Abcam), Nav1.3 1:200 (ASC-004, Alomone labs), Nav1.4 1:200 (ASC-020, Alomone labs), Nav1.5 1:200 (AGP-008, Alomone labs), Nav1.6 1:200 (ASC-009, Alomone labs), Nav1.7 1:200 (ASC-008, Alomone labs), Nav1.8 1:200 (AGP-029, Alomone labs), Nav1.9 1:200 (AGP-030, Alomone labs), Pan Nav (Nav) 1:200 (ASC-003, Alomone labs), and Zonula occludens-1 (ZO-1) 1:50 (33-9100, Life Technologies). All primary antibodies were diluted in 3% BSA in PBS and incubated for 1 h.
The incubation with primary antibodies was followed by three PBS washes and 1 h incubation with secondary antibodies; goat anti-rabbit Alexa Fluor 568 (A-11011), donkey anti-rabbit Alexa Fluor 488 (A-21206), donkey anti-mouse Alexa Fluor 568 (A10037), donkey anti-mouse Alexa Fluor 488 (A-21202), goat anti-guinea pig Alexa Fluor 568 (A-11075), goat anti-mouse Alexa Fluor 488 (A-11029), donkey anti-rabbit Alexa 647 (A-31573), donkey anti-mouse Alexa 647 (A-21236), goat anti-guinea pig Alexa Fluor 647 (A-21450) and goat anti-mouse Alexa Fluor 405 (A-31553) (all from Molecular Probes, Thermo Fisher Scientific) diluted 1:200 in 3% BSA in PBS. Actin was visualized using either a direct phalloidin Alexa Fluor 647 conjugate 1:50 (A22287, Thermo Fisher Scientific), Atto-633 1:50 (68825, Sigma-Aldrich) or tetramethylrhodamine B conjugate 1:400 (P1951, Sigma-Aldrich) and the nuclei were stained with 4′, 6′-diamidino-2-phenylidole (DAPI) included in the ProLong Gold antifade mounting medium (P36935, Thermo Fisher Scientific).
Pre-embedding immunogold labeling
The hESC-derived RPE monolayers were washed three times with phosphate-buffered saline (PBS) and then fixed for 2 h at RT in periodate-lysine-paraformaldehyde (PLP) fixative. Fixed cells were prepared for pre-embedding EM as described previously [76, 77]. Cells were treated with 0.01% saponin and 0.1% BSA in 0.1 M phosphate buffer, pH 7.4 (Buffer A) before adding the primary antibodies diluted in Buffer A. The concentration of all primary antibodies was doubled for the experiment compared to immunolabeling. After 1 h incubation at RT and washes with Buffer A, 1.4 nm nanogold-conjugated polyclonal Fab’ fragment of goat anti-rabbit IgG or of goat anti-mouse IgG (Nanoprobes.com, Yaphank, NY, USA) diluted to 1:50 in Buffer A was applied for 1 h, followed by washes with Buffer A and 0.1 M phosphate buffer (pH 7.4). Cells were post-fixed with 1% glutaraldehyde in phosphate buffer for 10 min at RT, quenched with 50 mM NH4Cl in phosphate buffer for 5 min at RT, and then washed with phosphate buffer and water.
The samples were treated in dark with HQ-silver (Nanoprobes.com) for 5 min followed by washes with water and gold toning (2% sodium acetate 3 × 5 min at RT, 0.05% gold chloride 10 min at +4 °C, 0.3% sodium thiosulphate 2 × 10 min at +4 °C). After washes with water, the cells were reduced in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at +4 °C and dehydrated with graded series of ethanol (70%, 96%, 100%), then stained with 2% uranyl acetate. Finally, the monolayers were embedded in Epon (TAAB Embedding resin, medium, TAAB Laboratories Equipment Ltd., Berks, UK) and after polymerization, sections perpendicular to the membrane were cut with an ultramicrotome (Leica ultracut UCT ultramicrotome, Leica Mikrosysteme GmbH, Austria). The thin sections (200 nm) were placed on carbon-coated single-slot grids and were imaged with JEOL JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) equipped with bottom-mounted Quemesa CCD camera (4008 × 2664 pixels). High voltage of 80 kV was used for imaging.
The hESC-derived RPE and ARPE-19 protein lysates were obtained by incubating 1 × 106 cell pellets in RIPA buffer supplemented with Halt protease inhibitor cocktail (87786, Thermo Fisher Scientific) for 30 min at +4 °C on constant agitation. The lysate was then centrifuged at +4 °C for 20 min at 12,000×g, mixed with Novex sample buffer (NP0007, Thermo Fisher Scientific) and heated at +70 °C for 10 min. The protein lysates were then loaded onto 3–8% NuPage gel (EA0375, Thermo Fisher Scientific) or Bolt™ 4–12% Bis-Tris Plus Gels (NW04120, Thermo Fisher Scientific), fractionated by SDS-PAGE and then either processed for MS analysis or transferred to nitrocellulose membrane via Trans Blot Turbo Transfer system according to the manufacturer’s protocols (BioRad).
The resulting blot was blocked with 3% BSA in PBS + 0.1% Tween-20 5 h at RT and then labeled overnight at +4 °C with the primary antibodies against various Nav subtypes diluted in blocking solution. The following antibodies were labeled with this protocol: Nav1.4 1:500 (PA5-36989, Thermo Fisher Scientific), Nav1.5 1:500 (AGP-008, Alomone labs), Nav1.6 1:1000 (ASC-009, Alomone labs), and β-actin 1:2000 (ab6276, Abcam). The membranes were subsequently washed three times for 15 min with PBS + 0.1% Tween-20 and incubated with a 1:20,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (ab6721, Abcam), goat anti-guinea pig IgG (ab6908, Abcam), or anti-mouse IgG (A-21236, Thermo Fisher Scientific) antibodies for 1 h at RT. For Nav1.8 1:5000 (ASC-016, Alomone labs), the protocol was modified as follows: blocking was overnight at +4 °C, primary antibody labeling was for 1 h at RT, washing was three times for 10 min with PBS + 0.01% Tween-20 and the secondary antibody was incubated with a 1:3000 dilution for 1 h at RT. After subsequent washes, the membranes were developed with the WesternBright ECL system (K-12045-D20, Advansta) and imaged with ChemiDoc XRS+.
Sample preparation of mass spectrometry
The SDS-page gels were labeled overnight at RT with coomassie blue dye to identify the bands. Protein bands ranging from 200 to 260 kDA were excised from the gel and destained by submerging the samples in acetonitrile (ACN) and 50 mM triethyl ammonium bicarbonate (TEAB) (1:1) solution for 30 min. Samples were subsequently alkylated and reduced by adding 25 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 50 mM TEAB (1:1) and set in thermos mixer at +60 °C with interval mixing for 1 h. After supernatant removal, the samples were submerged with 10× Iodine acetamide in 50 mM TEAB for 30 min in dark. Samples were then washed with 50 mM TEAB:ACN 1:1 solution three times and dried with vacuum concentrator prior to trypsinization (1 μg of trypsin in 50 mM ammonium bicarbonate solution) for 16 h at +37 °C. Obtained peptides were eluted from the gel fragments using 50% ACN, 5% formic acid (FA) solution. Supernatants were again dried using vacuum concentrator, eluted to the analysis buffer (2% Acetonitrile, 0.1% Formic acid), and injected to the NanoLC-MSTOF instrument. All solvents and other materials were purchased from Thermo Fisher Scientific (San Jose, CA, USA) except Trypsin (TPKC treated, Sciex).
Identification of proteins
Identification of the proteins was done using Protein Pilot® 4.5 (Sciex, Redwood City, USA) and all data dependent analysis (DDA) runs MS/MS spectra were identified against respective Nav channel protein data retrieved from UniprotKB/SwissProt library. FDR 1% and 99% peptide confidence level was used in the library creation and only distinctive peptides were used in the identification. Mass accuracy was set to 5 ppm for each peptide.
Proteins were analyzed by Nano-RPLC-MSTOF instrumentation using Eksigent 425 NanoLC coupled to high-speed TripleTOF™ 5600+ mass spectrometer (Ab Sciex, Concord, Canada). A microcapillary RP-LC column (cHiPLC® ChromXP C18-CL, 3 μm particle size, 120 Å, 75 μm i.d × 15 cm, Eksigent Concord, Canada) was used for LC separation of peptides. Samples were first loaded into trap column (cHiPLC® ChromXP C18-CL, 3 μm particle size, 120 Å, 75 μm i.d × 5 mm) from autosampler and flushed for 10 min at 2 μl/min (2% ACN, 0.1% FA). The flush system was then switched to line with analytical column. The peptide samples were analyzed with 120 min 6 step gradient using eluent A: 0.1% FA in 1% ACN and eluent B: 0.1% FA in ACN (eluent B from 5 to 7% over 2 min; 7 to 24% over 55 min; 24 to 40% over 29 min; 40 to 60% over 6 min; 60 to 90% over 2 min and kept at 90% for 15 min; 90 to 5% over 0.1 min and kept at 5% for 13 min) at 300 nl/min.
The following key parameters were applied for TripleTOF mass spectrometer in shotgun identification analysis: ion spray voltage floating (ISVF) 2300 V, curtain gas (CUR) 30, interface heater temperature (IHT) +125 °C, ion source gas 1 13, declustering potential (DP) 100 V. Methods were run by Analyst TF 1.5 software (Ab Sciex, USA). For IDA parameters, 0.25 s MS survey scan in the mass range 350–1250 mz was followed by 60 MS/MS scans in the mass range of 100–1500 Da (total cycle time 3.302 s). Switching criteria were set to ions greater than mass to charge ratio (m/z) 350 and smaller than 1250 (m/z) with charge state 2–5 and an abundance threshold of more than 120 counts. Former target ions were excluded for 12 S. IDA rolling 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 hESC-derived 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% CO2. In the blocker experiments, selective blockers for Nav1.4, Nav1.8 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 Nav1.4 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, signal-to-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  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).
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.
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.
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).
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
The authors declare that they have no competing interests.
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