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TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium

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TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium Retinal Cell Biology TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium Peter Y. Zhao,1 Geliang Gan,1 Shaomin Peng,1–3 Shao-Bin Wang,1,2 Bo Chen,2 Ron A. Adelman,2 and Lawrence J. Rizzolo1,2 1Department of Surgery, Yale University, New Haven, Connecticut, United States 2Department of Ophthalmology & Visual Science, Yale University, New Haven, Connecticut, United States 3Aier School of Ophthalmology, Central South University, Changsha, China Correspondence: Lawrence J. Rizzo- PURPOSE. Calcium regulates many functions of the RPE. Its concentration in the subretinal lo,DepartmentofSurgery,Yale space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are University, PO Box 208062, New a superfamily of ion channels that are moderately calcium-selective. This study investigates Haven, CT 06520-8062, USA; the subcellular localization and potential functions of TRP channels in a first-passage culture [email protected]. model of human fetal RPE (hfRPE). Submitted: September 24, 2014 Accepted: February 17, 2015 METHODS. The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum- free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence Citation: Zhao PY, Gan G, Peng S, et al. of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). TRP channels localize to subdomains The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, of the apical plasma membrane in human fetal retinal pigment epitheli- and immunofluorescence confocal microscopy. um. Invest Ophthalmol Vis Sci. RESULTS. Barium chloride substantially decreased TER and disrupted cell–cell contacts when 2015;56:1916–1923. DOI:10.1167/ added to the apical surface of RPE, but not when added to the basolateral surface. The effect iovs.14-15738 could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC- 067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. CONCLUSIONS. The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca2þ] in the subretinal space. Keywords: retinal pigment epithelium, TRP channels, tight junction, subretinal space he role of calcium in the subretinal space remains under junctions transmits extracellular signals that regulate various T investigation. The subretinal space lies between the cellular functions.4,5 photoreceptors and the RPE. The RPE is a monolayer of Polarity and barrier function allow the RPE to maintain the pigmented cuboidal cells in the eye that supports visual neural retina in a relatively dehydrated state. As the RPE pumps function and forms the outer blood–retinal barrier. The RPE chloride from the subretinal space into the choroid, cations and regulates the outer blood–retinal barrier, phagocytoses the water passively follow. Further, RPE buffers the ion composi- outer segment discs shed by photoreceptors, secretes growth tion of the subretinal space to compensate for shifts caused by factors, regenerates 11-cis-retinal, and absorbs stray light. Many changes in photoreceptor activity throughout the day-night 2þ functions of the RPE are segregated to apical (facing the cycle. Light induces a rapid decrease of Ca in the subretinal 2þ subretinal space) and basolateral (facing the choroid) domains space ([Ca ]SRS) followed by a slow increase in volume and efflux of Ca2þ from photoreceptors into the subretinal space.6,7 of the plasma membrane.1 The polarized distribution of The mechanisms by which RPE senses and regulates volumeSRS proteins between these domains is maintained by the fence 2þ and [Ca ]SRS are unknown. function of tight junctions, which encircle each cell in the 2þ Intracellular free calcium ([Ca ]i) acts as a secondary plane of the monolayer. The barrier function of tight junctions messenger and/or directly modifies protein structure to results from binding neighboring cells with a partially 2þ regulate many cell functions. In the RPE, Ca i is tightly occluding seal. This seal selectively retards diffusion between regulated and affects transport of ions and water, secretion of 2 the subretinal space and the fenestrated choriocapillaris. The growth factors, and phagocytosis of shed photoreceptor outer combination of polarity and barrier function allow RPE to segments.3,8–10 For example, extracellular ligands can increase 2þ 11 regulate transcellular transport, thereby regulating the ionic [Ca ]i to stimulate fluid transport. The RPE continuously milieu of the subretinal space. This combination allows RPE to extrudes Ca2þ from the cytosol, using pumps and exchangers to exchange retinal nutrients and metabolic waste with the move Ca2þ into the extracellular space, endoplasmic reticulum, choriocapillaris.3 In addition, the signaling function of the tight and melanosomes. Continuous efflux is opposed by influx Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 1916 Downloaded from tvst.arvojournals.org on 10/01/2021 TRP Channels of Human Fetal RPE IOVS j March 2015 j Vol. 56 j No. 3 j 1917 2þ through ion channels. The net effect is that [Ca ]i is on the day of the experiment (DMSO <1% of total media). maintained at approximately 100 nM, four orders of magnitude Caloxin 1b1 (Anaspec, Inc., Fremont, CA, USA) was dissolved below the extracellular concentration. There are local varia- in SFM-1. Final concentrations were: 3.0 mM BaCl2 (Thermo 2þ 12 tions in [Ca ]i, as exemplified by the primary cilium of RPE. Fisher Scientific, Rockford, IL, USA), 6.3 mM CaCl2 (Sigma- 2þ The resting [Ca ]cilia is approximately 7 times higher than Aldrich Corp., St. Louis, MO, USA), 2.0 mM LaCl3 (Sigma- 2þ resting [Ca ]cytoplasm, and regulation of hedgehog signaling Aldrich Corp.), 5.0 lM valinomycin (R&D Systems, Minneap- 2þ was effected by manipulating [Ca ]cilia without changing olis, MN, USA), 10 lM nifedipine (Alfa Aesar, Ward Hill, MA, 2þ [Ca ]cytoplasm. USA), 20 lM ML204 (Sigma-Aldrich Corp.), 1.0 lM HC-067047 In RPE, Ca2þ influx from the extracellular space is mediated (Sigma-Aldrich Corp.), 100 lM ALLM (Santa Cruz Biotechnol- by three families of ion channels at the plasma membrane: ogy, Dallas, TX, USA), and 400 lM caloxin 1b1. Solutions were voltage-dependent L-type channels, ligand-gated channels, and added to apical and basolateral media chambers unless transient receptor potential (TRP) channels.3 Closed at otherwise specified. After media exchange, tissue culture physiologic membrane potentials, voltage-dependent L-type plates were agitated gently to mix the media and incubated channels open when phosphorylated by tyrosine kinase.13 at 378C. The TER was measured before media exchange, and at Ligand-gated channels are activated by extracellular purinergic 2 and 4 hours of incubation. compounds.11 In contrast, TRP channels mediate the consti- 2þ 2þ tutive Ca influx that defines the resting [Ca ]i for ARPE19 Quantitative Real-Time Reverse-Transcription PCR 14 cells. The mammalian TRP channel superfamily is divided (qRT2-PCR) primarily into TRPC, TRPM, and TRPV subfamilies, though each individual subfamily member has diverse roles and Total RNA was extracted using the RNeasy Mini kit (Qiagen, mechanisms of activation. The specific TRP channels ex- Valencia, CA, USA). The cDNA was reverse transcribed from pressed in human RPE tissue are not well characterized. 2.0 lg total RNA using the QuantiTect Reverse Transcription To study TRP channels, we used a well-established culture Kit (Qiagen). Reactions were performed using iTaq SYBR model of human fetal RPE (hfRPE) isolated from 15- to 16-week Green (Bio-Rad, Hercules, CA, USA) and the primers listed in gestation fetal eyes and maintained in defined serum-free Supplementary Table S1. Relative mRNA expression was media.15 Cultures of hfRPE are highly polarized, as reflected by calculated using the 2ÀDDCt method, normalized to GAPDH a transepithelial electrical resistance (TER) > 300 X 3 cm2 and and CLDN19.19 All reactions were performed in triplicate from by functional and electrophysiological properties attributed to a minimum of two cell samples from different primary native adult human RPE.16,17 Using Ba2þ as a Ca2þ surrogate, cultures. we show there are apical pathways for entry of divalent cations. These pathways are active in the absence of external Immunoblotting stimulation and are blocked by the nonselective TRP channel inhibitor La3þ. We characterize the TRP channel expression Samples were washed with ice cold PBS and lysed by profile of hfRPE, and show the localization of TRPC4, TRPM3, sonication into a 25 mM Tris (pH 8.0) solution containing and TRPV4 to subdomains of the apical plasma membrane. protease inhibitor and 1% SDS. Then, 20 lg protein extracts were diluted in Laemmli sample buffer, separated on a 7.5% Tris-HCl polyacrylamide gel (Bio-Rad), and transferred to MATERIALS AND METHODS polyvinylidine fluoride (PVDF) membranes (PerkinElmer, Inc., Santa Clara, CA, USA). After 1 hour incubation in Membrane Cell Culture Blocking Solution (Life Technologies, Frederick, MD, USA), membranes were incubated with primary antibodies (Supple- Primary cultures of hfRPE were obtained from Sheldon Miller mentary Table S2) overnight at 48C. Membranes were washed (National Eye Institute). Research adhered to the tenets of the three times with PBS containing 0.2% Tween and incubated Declaration of Helsinki. Cultures were trypsinized and with horseradish peroxidase-conjugated donkey anti-rabbit reseeded on 0.4-lm pore Transwell or Snapwell culture inserts antibodies (1:3000, Thermo Fischer Scientific) for 1 hour.
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