TRP Channel Gene Expression in the Mouse Retina ⇑ Jared C

Vision Research 51 (2011) 2440–2452

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Vision Research

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TRP channel gene expression in the mouse retina ⇑ Jared C. Gilliam, Theodore G. Wensel

Verna and Marrs McLean, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, United States article info abstract

Article history: In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes Received 3 August 2011 were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by Received in revised form 3 October 2011 RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched Available online 20 October 2011 in the retina. The strongest signals were observed for TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1, and clear signals were obtained for TRPC4, TRPM7, TRPP2, TRPV2, and TRPV4. In situ hybridization and immu- Keywords: nofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, Retina TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed TRP channels for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 Gene expression Mouse in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal Ion channels in cone outer segments was observed for TRPM7 and TRPP2. TRPC5 immunostaining was largely confined to INL cells immediately adjacent to the inner plexiform layer. TRPV2 antibodies stained photoreceptor axons in the outer plexiform layer. Expression of TRPM1 splice variants was strong in the ciliary body, whereas TRPM3 was strongly expressed in the retinal pigmented epithelium. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction is divided among six subfamilies, TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV. TRP channels, which can exist as homo- or hetero-tetra- The mammalian retina contains more than fifty distinct types of meric assemblies, have been implicated in physiological responses neurons, each responding to physiological stimuli with changes in to many stimuli, including light, taste, temperature, pH, and osmo- their membrane potentials and intracellular ion concentrations larity in neurons as well as in non-neuronal tissue (Montell & Ru- (Dowling & Boycott, 1966; Masland & Raviola, 2000), as well as bin, 1989; Xu et al., 2000). Various stimuli, such as ligand binding, glial cells essential for neuronal function and health. Environmen- membrane-stretch, thermal heat, endocannabinoids and phospho- tal changes in light, intraocular pressure, oxidative stress, ion con- lipids, have been proposed as modulators of TRP channel currents centrations and transmitters signaling circadian rhythm are (for reviews see Damann, Voets, & Nilius, 2008; Hardie, 2007; Ram- continuously sampled for fluctuations, resulting in the opening sey, Delling, & Clapham, 2006; Talavera, Nilius, & Voets, 2008). and closing of ion channels and alterations in membrane polariza- Physiological functions of most TRP channels have yet to be tion (Aldebasi et al., 2004; Brzezinski et al., 2005; Li & Puro, 2002). determined, but there is evidence suggesting TRP channels are To maintain the precision of the visual system, a large number of important in the mammalian retina. TRP mRNAs and proteins have ion channels are necessary to regulate electrophysiology in retinal been reported in mammalian retinas (Da Silva et al., 2008; Kim neurons. Although many cation-selective currents have been iden- et al., 2008; Sekaran et al., 2007; Wissenbach et al., 1998; Yazulla tified in the retina, most of the channels responsible for membrane & Studholme, 2004) and epiretinal tissues (Kennedy et al., 2010), potential modulation have not been identified at the molecular le- TRPV1 (Sappington et al., 2009; Shen et al., 2009) and TRPV4 (Rysk- vel (Ke et al., 2009; Nawy, 2000). amp et al., 2011) have been reported to contribute to pressure-in- Members of the transient receptor potential (TRP) family of cat- duced apoptosis in retinal ganglion cells, mutations in TRPM1 ion channels in most cases display broad selectivity toward phys- cause night blindness (Audo et al., 2009; Li et al., 2009; van Gend- iological cations, and with a few exceptions, are permeable to eren et al., 2009), and TRPML1 mutations cause retinal degenera- calcium. The mammalian TRP superfamily contains 28 members, tion (Sun et al., 2000). TRP channels have been proposed to form each with a proposed topology of six transmembrane helices, and the transduction channels of intrinsically photo-sensitive ganglion cells (Sekaran et al., 2007; Warren et al., 2006). ⇑ In this study, we investigated the expression and relative Corresponding author. Address: Department of Biochemistry and Molecular abundance of mRNA encoding mammalian TRP channels in the Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, United States. Fax: +1 713 796 9438. retina using RT-PCR and northern blot analysis. Abundant E-mail address: [email protected] (T.G. Wensel). candidates were then evaluated for cell-type-specific enrichment

0042-6989/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.visres.2011.10.009 J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452 2441 using in situ hybridization (ISH). Where satisfactory antibodies continued for 24 h, followed by cryoprotection in 30% sucrose for could be obtained, TRP channel proteins were tentatively identified 12 h at 4 °C, embedding into OCT, and freezing on dry ice. Cryosec- by immunoblotting and localized by immunofluorescence. tions (20 lm) were collected on Superfrost plus slides, treated with 1 lg/ml proteinase K for 10 min, and acetylated with 6.61 lM ace- 2. Materials and methods tic anhydride in 0.1 M triethanolamine for 10 min before overnight hybridization with digoxigenin-labeled RNA probes at 65 °C. Sec- 2.1. Animals tions were washed with 1 SSC/50% formamide at 65 °C before treating with 20 lg/ml RNAse A at 37 °C, and subsequent washes Adult C57BL/6 (Jackson Laboratory, Bar Harbor, ME) and C57BL/ in 2 SSC and 0.2 SSC applied at 65 °C. Anti-digoxigenin conju- 6 albino (Harlan Laboratories, Indianapolis, IN) mice of both sexes gated alkaline phosphatase-labeled sections were developed with were used in this study. TRPM1/ mice were generated by Lexicon 5-bromo-4-chloro-3-indolyl phosphate/4-nitroblue tetrazolium Genetics (TRPM1tm1Lex) on a C57BL/6;129S5/SvEvBrd genetic back- chloride from 3 to 72 h at RT. Sections were extensively washed ground and obtained through the European Mouse Mutant Archive. in 1 PBS (pH 7.4), treated with 4% paraformaldehyde in 1 PBS All animals were handled according to NIH guidelines and EU for 30 min at RT, and coverslipped. Directive 2010/63/EU; all procedures used were approved by the Baylor College of Medicine Institutional Animal Care and Use 2.5. Buffers Committee. The composition of solutions were as follows – low salt buffer: 2.2. Reverse transcription of cDNA and PCR (RT-PCR) 5 mM Tris (pH 7.5), 5 mM EDTA, 5 mM DTT, solid PMSF; high salt buffer: 5 mM Tris (pH 7.5), 1 M NaCl, 2 mM EDTA; membrane buf- Retinas were homogenized in 4 M guanidinium isothiocyanate, fer: 5 mM Tris (pH 7.5), 20 mM NaCl, 2% SDS, 2 mM EDTA. All buf- 25 mM sodium citrate (pH 7.0), 0.5% N-lauroylsarcosine, 0.1 M 2- fers contained protease inhibitors (6 lg/mL aprotinin, 6 lg/mL mercaptoethanol, and passaged through a 23 Ga needle. Lysates chymostatin, 1.5 lg/mL leupeptin, 2.0 lg/mL pepstatin A, 0.9 mg/ were ultracentrifuged through 5.7 M CsCl at 175,000g for 18 h at mL trypsin inhibitor, 4.7 mg/mL benzamide, 1.2 lg/mL e-64, and 0.12 mg/mL pefabloc). 22 °C. RNA pellets were washed with 70% ethanol and resuspended in the homogenization solution minus 2-mercaptoethanol. Follow- ing three rounds of extraction using 24:1 chloroform:isoamyl alco- 2.6. Preparation and washing of membranes from mouse tissues hol, RNA was precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and two volumes of 100% ethanol at 20 °C. Pellets were Tissues were homogenized in low salt buffer on ice and total washed in 80% ethanol, air-dried at room temperature, and stored membranes were collected by centrifugation at 100,000g for at 80 °C. Purified RNA was reverse transcribed for RT-PCR using 60 min at 4 °C. Membranes were resuspended in high salt buffer the Superscript III reverse transcriptase (Invitrogen) and the man- by extrusion through an 18 Ga needle and sedimented at 100,000g ufacturer’s protocol. All RNA solutions were prepared using diethyl for 30 min at 4 °C. Membrane pellets were solubilized with mem- pyrocarbonate-treated water. Following quantification using brane buffer and brief sonication on ice followed by centrifugation Quant-IT OliGreen (Invitrogen), 100 ng of cDNA was used to ampli- at 100,000g for 15 min at 4 °C. Protein amounts in solubilized fy TRP channel sequences. membranes were quantified using bicinchoninic acid assay and then they were used for electrophoresis. 2.3. Northern blot analysis 2.7. Immunostaining Total RNA was isolated from multiple mouse tissues homogenized in cold TRI Reagent (Ambion). Fresh eyes were hemisected Retinas were carefully separated from the eyecup and fixed in at the ora serrata to remove the cornea; retinas were carefully 4% paraformaldehyde in Ringer’s buffer (10 mM HEPES (pH 7.4), peeled from RPE/eyecups to limit tissue contamination and imme- 130 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, diately frozen on dry ice. Frozen hearts were thoroughly disrupted 0.02 mM EDTA) for 3 h at 4 °C. Retinas were washed in Ringer’s prior to RNA isolation while frozen retinas and RPE/eyecups were buffer and embedded in 5% agarose in Ringer’s buffer prior to homogenized directly. RNA (20 lgor10lg for RPE/eyecup) was vibratome sectioning. Sections (150 lm) were pre-blocked for 6 h resolved using 1% agarose gels containing 0.25 M formaldehyde at 4 °C in 10% donkey serum, 5% BSA, 0.5% fish gelatin, 0.1% Triton and transferred overnight to BrightStar positively charged nylon X-100 in Ringer’s buffer. Primary antibodies were diluted in block- membranes (Ambion). Gene-specific cDNAs were quantified using ing solution and applied to tissue sections overnight at 4 °C. After Quant-IT PicoGreen (Invitrogen) and used to generate high specific three washes using Ringer’s buffer, Alexa 488 or 555 conjugated activity cDNA probes (>4.0 109 cpm/lg) by random-prime label- antibodies were applied to sections for 1 h at room temperature ing with [a-32P] dCTP (6000 Ci/mmol) using the DECAprime II kit in blocking solution. Confocal images were acquired using a Zeiss and hybridized at 107 cpm/mL for 15 h at 42 °C in ULTAhyb (Ambi- laser scanning microscope LSM 510 (Carl Zeiss Microimaging, Inc.). on). Washes using 2 SSC/0.1% SDS and 0.5 SSC/0.1% SDS were applied at 60 °C, followed by 0.1 SSC/0.1% SDS at 42 °C prior to 3. Results exposing membranes to phosphorscreens. Phosphorscreens were imaged on a Typhoon image scanner (GE Healthcare). 3.1. Expression of TRP channel mRNAs in the retina

2.4. In situ hybridization 3.1.1. RT-PCR of TRP channel sequences Initially, we used RT-PCR for highly sensitive TRP mRNA detec- Eyes were carefully dissected from the orbit following lid re- tion. PCR primers were designed to amplify across the intron/exon moval. Extraoccular muscles were severed with scissors to mini- boundaries of published TRP channel sequences (Table S1) (Bult mize retinal distortion. Following corneal puncture, eyes were et al., 2008). Amplified sequences were cloned and verified by DNA fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.0) sequencing (Fig. 1, Table 1). Results revealed that mRNA is present for 1 h at 4 °C. After removal of the cornea and lens, eyecup fixation in the retina from all 28 TRP channel genes as well as from the gene 2442 J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452

different distributions of relative abundance of each variant, and TRPM1 reveals strikingly distinct transcripts differentially expressed in the retina or the eyecup. Transcripts for the remaining channels gave very weak or indistinct bands. These low abundance RNA species may be present only in rare cell types, or simply expressed at very low levels in numerous cells. Because important functions have been attributed to TRPV1 in the retina (Sappington et al., 2009; Zimov & Yazulla, 2004, 2007), we checked whether the weakness of the signal we observed was due to the quality of the probe by using dorsal root ganglion (DRG), a known site of TRPV1 expression, as a positive control. The probe used reliably detected TRPV1 message in DRG (Fig. S2), leading to the conclusion that TRPV1 mRNA levels are in- deed quite low compared to those in DRG, and likely to those of other retinal TRP channels.

3.2. Localization of TRP channel mRNAs in the retina

To localize mRNA, ISH was carried out on sections from frozen retinas. Antisense probes for TRPC1, TRPC3, TRPC4, PKD1, and TRPP2 revealed expression of these genes in all layers of the neural retina. TRPC1 signal was especially strong in the proximal portion of the photoreceptor inner segments, corresponding to the ribo- some-rich myoid region (Fig. 3). The relative strength and non-uni- formity of distribution of some mRNAs is a consistent finding that was replicated in hybridizations using two different mRNA probes, with a minimum of three independent hybridizations used for each mRNA probe. Signal for TRPML1 was detected in the photoreceptors in the outer nuclear layer, and also the outer plexiform layer (OPL). This channel is thought to be predominantly present in lysosomal or other intracellular membranes, so its expression in photoreceptors Fig. 1. Detection of transient receptor potential gene transcripts from adult mouse is likely to be in internal membranes of the photoreceptors and of retina cDNA. RT-PCR analysis using 100 ng of cDNA yielded amplicons for all 28 TRP processes within the OPL. channels as well as PKD1, a TRP-related gene. Multiple splice variants were detected for some TRP channel genes. Expression of both TRPM1 and TRPM3 is limited to the inner nuclear layer (INL), but each signal is limited to a different popula- for polycystic kidney disease 1 (PKD1), a TRP-associated protein tion of cells. Message for TRPM1 occupied a row of neurons adja- known to bind TRPP2 (Qian et al., 1997; Tsiokas et al., 1997). Multi- cent to the OPL, consistent with its known expression in rod ple splice variants were detected for some TRP channels, including bipolar cells (Kim et al., 2008). Antisense probe for TRPM3 hybrid- TRPC1, TRPM3, TRPM5, TRPM8, TRPV1, and TRPML1. A second com- ized to a population of cells in the middle portion of the INL that plete set of PCR primers was designed to amplify sequences in the 30- appear distinct from rod bipolar cells. ISH using probes prepared untranslated regions (30 UTR) of TRP channel mRNA (Table S2). RT- from both sets of cDNA sequences failed to reliably detect tran- PCR results using this second set of primers were consistent with re- scripts for the remaining TRP channels, consistent with northern sults obtained from the first set of primers, confirming that mes- data suggesting their low abundance in the retina. sages from all TRP channel genes are present at readily detectable Based on our results suggesting high expression of TRPM1 and levels in the retina. Because the retinal pigment epithelium (RPE) TRPM3 in the eyecup, and the published expression of TRPM1 in is adjacent to photoreceptor outer segments, message from the melanocytes (Deeds, Cronin, & Duncan, 2000; Fang & Setaluri, RPE may be a minor contaminant in RNA of the isolated retina, but 2000), we evaluated the expression of TRPM1 and TRPM3 from al- the distinct pattern of splice variants observed for some TRP chan- bino mice. No significant difference was detected in the level of nels in RPE as compared to isolated retina (see Section 3.1.2.), sug- expression for TRPM1 in albino tissue (Fig. 4), whereas the appear- gests that such contamination is quite low. ance of a new splice variant of TRPM3 was detected in the retina of albino mice with an approximate length of 7 kb (Fig. 4). No expres- 3.1.2. Relative abundance of TRP mRNAs by northern blot sion of TRPM1 was detected in the cells of the RPE by ISH, but a Northern blots were used to assess the relative abundance of strong signal was detected in the ciliary body of the anterior eye TRP channel mRNA in retina as compared to other tissues (Fig. 2, segment (Fig. 4B–D) in addition to the expression in the INL. The Figs. S1 and S2, and Table 1). Total RNA was isolated from mouse expression of TRPM1 was further confirmed by immunodetection tissues and probed for TRP channel (and PKD1) messages using in both the INL and the ciliary body (Fig. 5). In contrast, TRPM3 the first set cDNAs described above as riboprobes. Transcripts with exhibits a strong expression in the RPE with a weaker signal in the strongest retinal signals included TRPC1, TRPC3, TRPM1, the ciliary body (Fig. 4F–H). TRPM3, and TRPML1 (Fig. 2). Bands for TRPC4, TRPC5, TRPM7, TRPP2, TRPV2, and TRPV4 were clearly visible. Several TRP channel 3.3. Immunodetection of TRP channel protein in the retina mRNAs were also detected in the eyecup at similar or lower levels as compared to the retina. In contrast, splice variants of TRPM1 and We tested for protein expression by immunoblots of proteins TRPM3 are expressed in the eyecup at concentrations higher than transferred from SDS PAGE gels of membranes from retina and those seen in the retina. In the case of TRPM3, identical transcripts other tissues, and by immunofluorescence staining of vibratome- appear to be present in both the retina and eyecup, although with sectioned agarose-embedded retinas. It is well known that many, J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452 2443

Table 1 Summary of results for expression of TRP channel mRNA and protein.

TRP RT- Spl. var. Northerns ISH IB IF channel PCR amp. TRPA1 + w – nt nt TRPC1 ++ 2 ++ > brain > kidney IS, (ONl, INL, IPL, 90 kDa, retina > brain nd OPL, some cells in GCL) TRPC2 ++++ w – nt nt TRPC3 +++ ++ brain heart Mod. throughout, 66 kDa; retina < brain; Throughout, esp. NFL, GCL, OPL, inner-most INL esp. ONL, INL, OPL 95 kDa in heart TRPC4 +++ + brain ONL, INL, GCL 115 kDa, retina < brain Throughout, esp. GCL, INL TRPC5 +++ + < brain – 85 kDa, retina < brain. Inner row of INL TRPC6 ++++ w – nt nt TRPC7 +++ w – nt nt TRPM1 +++++ Multiple ++ < EC others Very strong and Many cross-reactive bands,; Weak non-speciﬁc staining throughout (present in KO) speciﬁc for INL, does not recognize strong in INL (only in WT), soma > dendrites, ciliary ciliary body recombinant protein body (only in WT) TRPM2 ++++ w – nt nt TRPM3 +++++ 2 ++ < EC brain INL, RPE (ONL, GCL) nt nt TRPM4 ++++ w – nt nt TRPM5 ++ 3 ? – nt nt TRPM6 + w – nt nt TRPM7 ++++ + EC, – 120 kDa, brain > retina Cone outer segments brain < kidney TRPM8 + ? – nt nt TRPML1 ++++ 3 ++ brain, OPL, ONL, (INL, nt nt kidney > EC GCL) TRPML2 ++ ? – nt nt TRPML3 ++++ w, EC – nt nt PKD1 +++ ++, ONL, INL, GCL nt nt smear EC others TRPP2 +++++ + EC, INL, GCL (ONL for Kidney > liver > Retina brain; Multiple layers; strongest in cone outer segments brain kidney both sense and bands match recombinant antisense) TRPP2 TRPP3 +++ w, EC – nt nt TRPP5 +++ w – nt nt TRPV1 ++++ 3–4 w; diff size m DRG – nt nt TRPV2 ++++ ++ EC, – 80 kDa doublet matches OPL some adjacent to but distinct from ribeye staining; heart < brain recombinant TRPV2; some cells in IPL brain > retina TRPV3 ++ w – Many cross-reactive bands Müller glia cells TRPV4 +++++ + EC, brain, –ntnt heart kidney TRPV5 + w – nt nt TRPV6 + w, EC – nt nt

RT-PCR: Number of + signs indicates relative intensity of bands (note that these do not necessarily correlate with relative message abundance under our conditions). Splice var.: number of splice variants detected by RT-PCR and sequencing; North. (Northern blots): Number of + signs indicates relative intensity of bands; w, very weak or undetectable; EC, clearly present in eyecup; ?, signal without clear band. ISH (In situ hybridization): –, signal not detectable above sense control levels; parentheses, weaker signals; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; RPE, retinal pigmented epithelium. IB (immunoblots): nt, not tested (due to lack of suitable antibodies); > and indicate relative signal strengths in different tissues. IF (immunofluorescence): NFL, nerve fiber layer. if not most, commercially available antibodies raised against pep- TRPC1 (Fig. 6A and B) antibodies reliably detected the b-isoform tides from mammalian TRP channels do not give reliable results of hTRPC1 expressed in yeast (80 kDa) as a positive control due to cross-reactivity comparable to or exceeding reactivity with (Fig. 6B) (Ong et al., 2002), and also detected a major band of the the intended TRP target. We tested many such antibodies, and correct size for TRPC1 in both the brain and the retina (93 kDa). found most of them unsatisfactory. Results presented here are lim- Consistent with mRNA levels of TRPC1 detected by northern blot- ited to those that meet one or more of the following criteria (see ting, TRPC1 protein levels appear higher in the retina than in the Table 2 and Table S3): (1) immunoblots indicated a small number brain. The same antibody was not able to detect TRPC1 reliably of bands, including ones whose mobilities were consistent with the in retinal neurons by immunofluorescence. expected protein size (or co-migrating with recombinant protein The highest levels of immunoreactivity for TRPC5 antibodies expressed in yeast), with relative intensities in different tissues were found in membranes of the brain and the retina. A protein qualitatively consistent with Northern results; (2) absence of at with an apparent molecular weight of 97 kDa was detected in least some distinguishable part of the signal in knockout animals both tissues, while a second band 75 kDa was detected only in (for TRPM1); (3) agreement on specific localization by immunoflu- the brain (Fig. 6D). At longer exposures to enhanced chemilumi- orescence with two different antibodies directed against different nescence, a 110 kDa protein, the expected size for full length epitopes; (4) consistency between ISH and immunofluorescence TRPC5, is also detected in brain and retina membranes. TRPC5 results. mRNA has been shown to exist as different sized variants (Okada A TRPM1 antibody described previously (Morgans et al., 2009) et al., 1998) that may be translated into TRPC5 variants of reduced detected TRPM1 (Fig. 5) in the INL and OPL, consistent with expres- molecular weight. In addition, a recent publication reported that sion in ON-bipolar cells. It also recognized TRPM1 in the ciliary endogenous TRPC5 from brain lysates migrate lower (97 kDa) body, consistent with results from ISH and northern blots. than expressed protein (Bezzerides et al., 2004). Taken together, 2444 J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452

Fig. 2. Northern blot analysis of TRP channel mRNA expression in multiple tissues from wildtype mice. Total RNA, 20 lg per tissue or 10 lg for eyecup, was probed with 25 ng radiolabeled TRP channel cDNA probes. Ethidium bromide stained 18S ribosomal bands are shown for each gel as a loading control. these facts may explain why this antibody, which has been verified nofluorescence detected weak immunoreactivity in all layers of in the TRPC5 KO mouse (manufacturer’s datasheet), identifies the retina, but a single row of neurons in the INL of the retina immunoreactive bands with apparent molecular weights other showed enhanced TRPC5 immunstaining (Fig. 6G and Fig. S3). This than 110 kDa. Use of this same monoclonal antibody for immu- row of neurons, possibly amacrine cells (positive identification J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452 2445

Fig. 3. Distribution of TRP channel expression in the wildtype mouse retina by in situ hybridization. Sections were hybridized with digoxigenin-labeled riboprobes against either non-coding sense control sequences (A, C, E, G, I, K, M, and O) or specific antisense mRNA sequences (B, D, F, H, J, L, N, and P) for individual TRP channels. Blue precipitate formed specifically in antisense sections indicates localization of TRP channel mRNA. ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bars = 50 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) remains to be confirmed with selective markers) did not display TRPM7 mRNA expression profile. It was previously shown that detectable TRPC5 mRNA by ISH, consistent with evidence that expression of TRPM7 in cell lines can yield protein at the molecular TRPC5 mRNA is not abundant within the retina. weights recognized by our antibody (Runnels, Yue, & Clapham, A TRPM7 antibody detected a protein of 200 kDa (the ex- 2001). Lower molecular weight bands may represent cross-reactiv- pected molecular weight for full length TRPM7) in the brain and ity or proteolysis of TRPM7. When used for tissue staining, this the retina, as well as protein at 160 kDa that was seen in the antibody detected a specific signal concentrated in the outer seg- brain, retina, and the heart (Fig. 7A). An additional 120 kDa pro- ments of cone photoreceptors (Fig. 7D and E). Co-labeling of retinal tein was recognized in all tissues tested and is consistent with the slices, using two different anti-TRPM7 antibodies, confirmed the 2446 J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452

specific localization of TRPM7 immunostaining to cone outer segments (Fig. 7G–J). Expression of TRPM7 primarily in a relatively rare cell type (in mice) could explain the lack of mRNA detection by ISH. TRPP2 antibodies were used to test its immunoreactivity in membrane fractions as well as in retinal sections. Consistent with mRNA levels, the antibody detected a TRPP2-sized protein in membrane fractions of all tissues tested as well as recombinant human TRPP2 expressed in yeast as a positive control (Fig. 7K). A single protein, larger than 100 kDa, was detected in all tissues as well as two additional, smaller bands that were detected in the kidney. Lower molecular weight bands detected in the brain and the optic nerve (Fig. 7K and L) are proteolysis fragments of TRPP2 that disap- peared with increasing concentrations of protease inhibitors in the homogenization buffers. However, high concentration of inhibitors confounded accurate quantification of tissue membranes, so these higher inhibitor concentrations needed to fully inhibit TRPP2 proteolysis were not used for immunoblotting. The identification of the recognized proteins as TRPP2 isoforms in the kidney was supported by results with a second antibody against TRPP2 that detected all of the same bands with the same relative mobilities. Immunofluorescence of retinal sections was carried out using the first antibody, revealing expression of TRPP2 in all layers of the neural retina, with especially bright staining of the cone outer segments, the INL, and the ganglion cell layer (Fig. 7L and O). Ganglion cell immunoreactivity includes the axons, as the optic nerve gave a robust TRPP2 band in immunoblots (Fig. 7L). Isolated rod outer segments were co-labeled with TRPP2 and tubulin antibodies to determine if TRPP2 was localized in the connecting cilium. The results were consistent with TRPP2 localization to cone outer segments and photoreceptor inner segments, but TRPP2 was not restricted to photoreceptor connecting cilia. Among mouse tissues, TRPV2 was highly enriched in the membrane fractions of the brain and the retina when tested using an antibody that correctly identified rTRPV2 (86 kDa) expressed in yeast (Fig. 8A), consistent with Northern results. TRPV2 staining localized to the OPL in retinal sections (Fig. 8). Because the OPL contains synaptic connections between the photoreceptors and inner retinal neurons, we used co-labeling of ribeye, a photoreceptor synapse marker, to narrow down the localization of TRPV2 immunoreactivity. When stained together with ribeye, TRPV2 staining does not colocalize with ribeye but appears to be located at a region of the photoreceptor axons adjacent to the ribbon synapses (Fig. 8F–I).

4. Discussion

The results presented here provide extensive new information concerning TRP channel expression in the retina, and suggest new directions for uncovering their functions. It is somewhat sur- prising that mRNA from every TRP channel gene is detectable in the mouse retina. Of course, the technique used, RT-PCR is extre- mely sensitive, and the presence of a small amount of mRNA does Fig. 4. Expression of TRPM1 and TRPM3 mRNA in the retina, RPE, and ciliary body not guarantee that a functionally important amount of protein is of albino mice. (A) Northern blot of TRPM1 in pigmented and albino mouse tissues. present. Moreover, the RT-PCR product intensities cannot be reli- Equivalent amounts of total RNA, 20 lg per tissue or 10 lg for eyecup, isolated from C57BL/6 or C57BL/6 albino mouse tissues. Ethidium bromide stained 18S ribosomal ably correlated with the levels of message present in the retina. RNA control is shown. (B) Localization of TRPM1 mRNA in the albino retina. Thus, the results imply that it will be worthwhile to check knock- RPE = retina pigmented epithelium, ONL = outer nuclear layer, INL = inner nuclear outs of every TRP channel gene for effects on retinal function and layer, GCL = ganglion cell layer. Localization of either (C) TRPM1 sense control health, and considering them as candidate genes for hereditary ret- riboprobe or (D) TRPM1 antisense riboprobe in the ciliary body of albino mice. (E) inal disease. Northern blot of TRPM3 in pigmented and albino mouse tissues. Equivalent amounts of total RNA, 20 lg per tissue or 10 lg for eyecup, isolated from C57BL/6 or At the mRNA level, the TRP family members fall into three main C57BL/6 albino mouse tissues. Ethidium bromide stained 18S ribosomal RNA groups. One has the highest levels of mRNA as indicated by north- control is shown. (F) Localization of TRPM3 mRNA in the albino retina and RPE. ern blots, and includes TRPC1, TRPC3, TRPC4, TRPM1, TRPM3, Localization of either (G) TRPM3 sense control riboprobe or (H) TRPM3 antisense TRPML1, TRPV2, and TRPV4. A second group has weaker but still riboprobe in the ciliary body of albino mice. Scale bars = 50 lm. robust expression, and includes TRPC4, TRPC5, TRPM7, and TRPP2. J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452 2447

Fig. 5. TRPM1 immunofluorescence is detected in the inner nuclear layer and the ciliary body of P14 day old mouse eyes. (A) DIC and (B) immunofluorescence in TRPM1/ compared to the (C and D) immunofluorescence in C57BL/6 wildtype retinas using rabbit anti-TRPM1 antibody at (C) 40 magnification and (D) 63 magnification. Immunostaining for TRPM1 as seen by (E) DIC and (F) immunofluorescence in the ciliary bodies of TRPM1/ mice compared to the (G) DIC and (H) immunofluorescence of C57BL/6 wildtype mouse staining. OS = outer segment, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bars = 50 lm.

Table 2 List of antibodies used.

Antibody Immunogen Source Host Dilution CtBP2/Ribeye Synthetic peptide: amino acids 361–445 BD Biosciences, 612044 Mouse 0.250 lg/mL (IF) of mouse CtBP2 TRPC1 Cytoplasmic N-terminal domain of human L. Tsiokas, OUHSC, (Oklahoma City, OK), clone 1F1 Mouse 1.5 lg/mL (IB) TRPC1 TRPC3 Synthetic peptide: 14 amino acids at ProSci Inc. (Poway, CA), 3905 Rabbit 2.0 lg/mL (IB), 5.0 lg/mL (IF) C-terminus TRPC5 Synthetic peptide: amino acids 827–845 NIH NeuroMab (Davis, CA) 75–104 Mouse 2.0 lg/mL (IB), 5.0 lg/mL (IF) (SKAESSSKRSFMGPSLKKL) of human TRPC5 TRPM1 Synthetic peptide Sigma (St. Louis, MO), HPA014785 Rabbit 2.0 lg/mL (IF) TRPM7 Synthetic peptide ProSci Inc. (Poway, CA), 46–531 Goat 2.0 lg/mL (IB), 5.0 lg/mL (IF) TRPM7 Synthetic peptide: amino acids 1817– NIH NeuroMab (Davis, CA) 75–114 Mouse 5.0 lg/mL (IF) 1863 at C-terminus of mouse TRPM7 TRPV2 Synthetic peptide: N-terminus of human Santa Cruz Biotechnology (Santa Cruz, CA), SC22520 Goat 1.0 lg/mL (IB), 5.0 lg/mL (IF) TRPV2 TRPP2 Synthetic peptide: amino acids 689–968 Santa Cruz Biotechnology (Santa Cruz, CA), SC25749 Rabbit 1.0 lg/mL (IB), 2.0 lg/mL (IF) at C-terminus of human TRPP2

IF, immunoﬂuorescence; IB, immunoblot.

Expression of the other 16 TRP channel genes was much weaker. from differences in protocol for ISH. The high stringency methods The high expression levels motivate pursuing further the potential we used may have decreased possible signal from low-abundance functions of the 12 most abundant species. However, the less TRPC6 mRNA. We tested for TRPC6 protein by immunoblotting abundant mRNA species could be functionally important. with two anti-TRPC6 antibodies and they were able to detect TRPC6 and TRPC7 mRNAs seem to be present at very low levels, TRPC6 protein only in the brain, but not in the retina. At longer but they have been proposed as candidates for the transduction exposures, some signal at a size corresponding to TRPC6 signal also channel of melanopsin-expressing retinal ganglion cells (Sekaran appeared in the retina lane, but at those exposures the antibody et al., 2007). In addition, a recent publication identiﬁed TRPC6 was cross-reactive with multiple proteins at sizes not expected within cells of the INL and the GCL of the retina (Wang et al., for TRPC6. Immunostaining of the retina with these antibodies 2011). We did not detect any TRPC6 mRNA in the retina by ISH, yielded results consistent with the work by Wang et al. Although and northern blotting indicates that TRPC6 mRNA levels are low the antibodies gave weak signal-to-background levels, slightly in the retina. The differences between our study and those of Wang stronger signal was detected in the lower row of cells in the INL et al. may be due in part to probe differences, but may also result as well as in the GCL. 2448 J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452

Fig. 6. Immunoreactivity of antibodies for TRPC proteins. (A and B), TRPC1 immunoblots from multiple mouse total membrane fractions incubated with secondary HRP- conjugated goat anti-mouse IgG without (A) and with (B) prior reaction with primary antibody. Immunoblot detects a band the size of mouse TRPC1, approximately 92 kDa, in brain and retina, and hTRPC1, approximately 80 kDa, expressed in yeast membranes. (C–G), TRPC5 immunoreactivity in mouse membranes and immunolocalization within the retina. (C) Secondary HRP-conjugated goat anti-mouse IgG immunoreactivity in mouse tissue membranes in the absence of primary antibody. (D) Detection of TRPC5- immunoreactive bands, approximately 85 kDa and 75 kDa, in the brain and retina using mouse anti-TRPC5 primary antibody. (E–G), TRPC5 antibody immunoﬂuorescence of retinal slices. (E) DIC (differential interference contrast) image; (F) staining by donkey anti-mouse Alexa 488 secondary antibody (no primary antibody) (G) mouse anti-TRPC5 antibody localization using the same secondary after incubation with TRPC5 primary antibody. Scale bars = 50 lm.

TRPV1 is barely detectable in the retina using a probe that reli- The most striking pattern of localization of both mRNA and ably detects TRPV1 message in dorsal root ganglion, yet TRPV1 has immunoreactivity was observed for TRPM1, which is now well been reported to play an important role in Ca2+ entry leading to established as essential for light responses of ON bipolar cells apoptosis in retinal ganglion cells under pressure (Sappington (Shen et al., 2009). As seen previously (Kim et al., 2008; Koike et al., 2009). Sappington et al. used an mRNA probe corresponding et al., 2010; Morgans et al., 2009; Nakajima et al., 2009), the stain- to an N-terminal portion of the expressed protein, whereas we ing and mRNA localization in the retina was consistent with used two probes, corresponding to a C-terminal region of the pro- expression in these neurons, but most of the staining was in the tein as well as the 50 UTR of the TRPV1 mRNA. Both probes failed to cell body, not the dendritic tips where mGluR6 is located. A new show significant signal by ISH of retina cryosections. Paraffin sec- finding is that TRPM1 is also expressed at even higher levels in tion ISH, used by Sappington et al., has inherent differences com- the ciliary body, and that the distribution of splice variants there pared with our ISH method and may have provided for a more is distinctly different from that in the retina. Complimentary obser- stable signal for low-abundance TRPV1 mRNA. We utilized an vations were made for the closest relative of TRPM1, TRPM3, which anti-TRPV1 antibody and tested it against yeast expressed rTRPV1. is also expressed strongly in the INL, with weaker mRNA signal in While our antibody detected the expressed protein, it failed to the outer nuclear layer and ganglion cell layer, but very strong detect significant levels of protein in brain or retina, and failed to expression in the retinal pigmented epithelium. Prior to its provide signal by immunostaining of the retina. The antibody we implication in ON-bipolar cell signaling, interest in TRPM1 cen- utilized, though specific for TRPV1, may not have been sensitive tered on its role in pigmented cells and melanoma (Duncan et al., enough to replicate results from the previous report. 1998; Fang & Setaluri, 2000; Miller et al., 2004; Zimov & Yazulla, J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452 2449

Fig. 7. Immunoreactivity of TRPM7 and TRPP2 antibodies in membranes and immunolocalization within the retina. (A) Detection of TRPM7 immunoreactivity in mouse membranes using goat anti-TRPM7 primary antibody. Immunofluorescence of retinal slices shows (B) DIC micrograph image (C) donkey anti-goat Alexa 488 secondary antibody (D) goat anti-TRPM7 antibody localization and (E) DIC and TRPM7 overlapped. OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, GCL = ganglion cell layer, DIC = differential interference contrast. Immunofluorescence imaging of co-labeled retina slices shows (F) donkey anti-mouse Alexa 555 and donkey anti-goat Alexa 488 (G) mouse anti-TRPM7 (H) goat anti-TRPM7 (I) overlapped imaged and (J) digital zoom of overlapped images. (K and L) Detection of TRPP2 immunoreactivity in mouse membranes and hTRPP2 expressed in yeast using identical rabbit anti-TRPP2 antibodies. ON = optic nerve. Immunofluorescence of retinal slices shows (M) donkey anti-rabbit Alexa 488 secondary antibody (N) rabbit anti-TRPP2 antibody localization (O) DIC overlaid with anti- TRPP2 immunofluorescence in cone OS and the OPL. Scale bars in (J) and (O) = 10 lm, all other scale bars = 50 lm.

2004), so it is notable that the pigmented ciliary body contains and it may play such a role in cone outer segments. TRPM7 is a high levels, suggesting a functional role related to pigmentation. bifunctional channel that could affect cone photoreceptor physiol- A relationship between TRPM3 function and pigmentation is sug- ogy through its channel activity or its C-terminal alpha kinase gested by our observation that albino animals show altered levels activity. Little is known about the endogenous activity and targets of expression of some splice variants as compared to pigmented of TRPM7 kinase activity, but this activity may be sensitive to mice. changes in the concentrations of intracellular divalent cations Decreased expression of shorter TRPM1 variants has been corre- (Penner & Fleig, 2007). Because TRPM7 is outwardly rectifying, it lated with an increase in metastatic melanomas in human pig- exhibits high divalent selectivity with little voltage-dependence mented melanocytes (Duncan et al., 2001; Fang & Setaluri, 2000). over the physiological voltage range for cone photoreceptors. Our discovery that variants of TRPM1 are differentially expressed While TRPM7 current is typically small under physiological con- at high levels in either the ciliary body or in the INL of the retina, centrations of extracellular cations, it is possible that TRPM7 mod- suggest the interesting possibility that TRPM1 could play a role ulates the intracellular concentration of Zn2+,Mg2+,Mn2+, and Co2+ in some forms of uveal melanoma and/or forms of melanoma-asso- as the intracellular concentration of Ca2+ changes with light- ciated retinopathy (MAR) that show selective reduction of b-wave dependent hyperpolarization. It is also possible that TRPM7 may amplitudes in electroretinogram (ERG) waveforms (Keltner, have some function in synaptic vesicle release in cone photorecep- Thirkill, & Yip, 2001; Kim, Alexander, & Fishman, 2008). tors (Krapivinsky et al., 2006). Another striking expression pattern is that of TRPM7, which two TRPP2 immunostaining is also enriched in cone outer segments, antibodies localize to cone outer segments. TRPM7 has been sug- which are modiﬁed cilia, consistent with its previous localization gested to be important in homeostasis of Mg2+ or other metal ions, to cilia in the kidney. While TRPP2 is often localized to primary ci- 2450 J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452

Fig. 8. Immunoreactivity of TRPV2 protein in membranes and immunolocalization within the retina. (A) Detection of TRPV2 immunoreactivity in mouse membranes and in yeast expressing recombinant rTRPV2 using goat anti-TRPV2 primary antibody. Immunofluorescence of retinal slices shows (B) DIC micrograph image (C) donkey anti-goat Alexa 488 secondary antibody (D) goat anti-TRPV2 antibody localization and (E) DIC and TRPV2 overlapped. OS = outer segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, GCL = ganglion cell layer, DIC = differential interference contrast. Co-staining using ribbon-synapse specific marker shows (F) mouse anti-Ribeye Alexa 555 (G) goat anti-TRPV2 Alexa 488 (H) overlapped images (I) digitally zoomed image of the OPL. Scale bars in (B–H) = 50 lm, scale bar in (I) = 50 lm. lia, it is not exclusively expressed in ciliary membranes (Hoffmei- with TRPC1 in the brain (Goel, Sinkins, & Schilling, 2002; Stru- ster et al., 2011), and was not restricted to the connecting cilia of bing et al., 2001). Though little is known regarding the function dissociated outer segments. TRPP2 is also expressed in other neu- of TRPC5 in neurons, the function of TRPC5 in the retina may be rons throughout the retina, especially in the INL, and may play a analogous to its function in the brain. role in the cilia of those cells, or in some unknown function. The TRPV2 also has a striking pattern of localization. It has previ- mRNA for PKD1, a known binding partner for TRPP2, is also de- ously been reported that TRPV2 is localized to the plexiform lay- tected in all nuclear layers, consistent with their forming a com- ers of the retina (Leonelli et al., 2009; Yazulla & Studholme, plex as they do in the kidney. Hydrostatic pressure and fluid flow 2004), and our data also demonstrate strong outer plexiform are known to be important for retinal health and it may be that this staining using an anti-TRPV2 antibody in 21 day old mice. This complex plays a sensing role similar to its role in the kidney. While staining appears to be localized to the photoreceptor axons, based TRPP2 has not been previously reported in the retina (please note on its proximity to pre-synaptic ribeye staining. However, we did that TRPP2 as used here is identical with the product of the PKD2 not detect TRPV2 staining in the IPL as has been reported previ- gene or polycystin-2, and is not to be confused with TRPP3/PKD2L, ously. Both previous studies utilized the same commercial TRPV2 which some authors refer to as TRPP2, while referring to TRPP2 as antibody, but neither validated the specificity of the antibody for ‘‘TRPP1’’) mutations in TRPP2 may lead to retinal damage (Feng TRPV2. The antibody we use was confirmed to recognize authen- et al., 2009). A recent publication has demonstrated that TRPP2 tic rTRPV2 expressed in yeast membranes, whose size and iden- can interact with retinitis pigmentosa protein RP2 suggesting a pos- tity were further confirmed via an epitope tag expressed at the sible link between TRPP2 and retinal ciliopathies (Hurd et al., rTRPV2 terminus. Leonelli et al. (2009) observed sparse immuno- 2010). staining in the IPL and GCL in the rat retina at P60, but not at P15, TRPC1 has been reported to interact with TRPP2 (Bai et al., so that even if the signal was really due to TRPV2, it may be that 2008; Tsiokas et al., 1999), and its expression levels and distri- P21 mouse retina does not contain detectable levels of TRPV2 in bution levels are such that it could easily do so in the retina. those cell layers. We did not detect TRPV2 immunoreactivity in However, its levels as indicated by both mRNA and immuno- mouse RGC neurons as was previously reported, but we cannot staining appear much higher relative to brain and kidney than rule out the possibility that at the age examined the levels of do the levels of TRPP2, suggesting that most of the TRPC1 likely TRPV2 is present in those cells but at levels below our detection has a function independent of that complex. One such function limit. In addition to the retina, TRPV2 is localized to the RPE, con- of TRPC1 could include modulating the Ca2+ concentration asso- sistent with our detection of TRPV2 mRNA in the eyecup, and ciated with tonic synaptic activity in photoreceptors (Szikra may play a role in thermal regulation of RPE cells (Cordeiro et al., 2008). TRPC1, TRPC3, TRPC4 and TRPC5 are all expressed et al., 2010). Although it has been demonstrated that TRPV2 is robustly in the retina, and, except for TRPC5, are found in multi- heat-sensitive (Caterina et al., 1999) additional functional roles ple cell types, as they are in other tissues, where many functions have been proposed, and it will be interesting to explore retinal have been proposed, but not yet unambiguously demonstrated. phenotypes of TRPV2 knockout mice. A common theme may be coupling to activation of PLC-linked Determining the levels and localization of TRP channel gene GPCR signaling cascades, which are numerous in retinal neurons. expression is only a first step toward elucidating their roles in ret- The TRPC5 staining of cells whose location suggests they may be inal function, health and disease. However, having the results of amacrine cells, could reflect a function specific for those cells, such a survey should prove a valuable tool for guiding future work whose identity will be important to test. TRPC5 is found in hip- on TRP channel function in the retina as well in less accessible pocampal neurons and has been shown to interact physically parts of the nervous system. J.C. Gilliam, T.G. Wensel / Vision Research 51 (2011) 2440–2452 2451

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