TRP channel gene expression in the mouse retina

Vision Research 51 (2011) 2440–2452

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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

a r t i c l e

i n f o

Article history: Received 3 August 2011 Received in revised form 3 October 2011 Available online 20 October 2011 Keywords: Retina TRP channels Gene expression Mouse Ion channels

a b s t r a c t In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched 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 immunofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal 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 The mammalian retina contains more than fifty distinct types of neurons, each responding to physiological stimuli with changes in their membrane potentials and intracellular ion concentrations (Dowling & Boycott, 1966; Masland & Raviola, 2000), as well as glial cells essential for neuronal function and health. Environmental changes in light, intraocular pressure, oxidative stress, ion concentrations and transmitters signaling circadian rhythm are continuously sampled for fluctuations, resulting in the opening and closing of ion channels and alterations in membrane polarization (Aldebasi et al., 2004; Brzezinski et al., 2005; Li & Puro, 2002). To maintain the precision of the visual system, a large number of ion channels are necessary to regulate electrophysiology in retinal neurons. Although many cation-selective currents have been identified in the retina, most of the channels responsible for membrane potential modulation have not been identified at the molecular level (Ke et al., 2009; Nawy, 2000). Members of the transient receptor potential (TRP) family of cation channels in most cases display broad selectivity toward physiological cations, and with a few exceptions, are permeable to calcium. The mammalian TRP superfamily contains 28 members, each with a proposed topology of six transmembrane helices, and ⇑ Corresponding author. Address: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, United States. Fax: +1 713 796 9438. E-mail address: [email protected] (T.G. Wensel). 0042-6989/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.visres.2011.10.009

is divided among six subfamilies, TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV. TRP channels, which can exist as homo- or hetero-tetrameric assemblies, have been implicated in physiological responses to many stimuli, including light, taste, temperature, pH, and osmolarity in neurons as well as in non-neuronal tissue (Montell & Rubin, 1989; Xu et al., 2000). Various stimuli, such as ligand binding, membrane-stretch, thermal heat, endocannabinoids and phospholipids, have been proposed as modulators of TRP channel currents (for reviews see Damann, Voets, & Nilius, 2008; Hardie, 2007; Ramsey, Delling, & Clapham, 2006; Talavera, Nilius, & Voets, 2008). Physiological functions of most TRP channels have yet to be determined, but there is evidence suggesting TRP channels are important in the mammalian retina. TRP mRNAs and proteins have been reported in mammalian retinas (Da Silva et al., 2008; Kim et al., 2008; Sekaran et al., 2007; Wissenbach et al., 1998; Yazulla & Studholme, 2004) and epiretinal tissues (Kennedy et al., 2010), TRPV1 (Sappington et al., 2009; Shen et al., 2009) and TRPV4 (Ryskamp et al., 2011) have been reported to contribute to pressure-induced apoptosis in retinal ganglion cells, mutations in TRPM1 cause night blindness (Audo et al., 2009; Li et al., 2009; van Genderen et al., 2009), and TRPML1 mutations cause retinal degeneration (Sun et al., 2000). TRP channels have been proposed to form 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 abundance of mRNA encoding mammalian TRP channels in the retina using RT-PCR and northern blot analysis. Abundant candidates were then evaluated for cell-type-specific enrichment

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using in situ hybridization (ISH). Where satisfactory antibodies could be obtained, TRP channel proteins were tentatively identified by immunoblotting and localized by immunofluorescence. 2. Materials and methods 2.1. Animals Adult C57BL/6 (Jackson Laboratory, Bar Harbor, ME) and C57BL/ 6 albino (Harlan Laboratories, Indianapolis, IN) mice of both sexes were used in this study. TRPM1/ mice were generated by Lexicon Genetics (TRPM1tm1Lex) on a C57BL/6;129S5/SvEvBrd genetic background and obtained through the European Mouse Mutant Archive. All animals were handled according to NIH guidelines and EU Directive 2010/63/EU; all procedures used were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. 2.2. Reverse transcription of cDNA and PCR (RT-PCR) Retinas were homogenized in 4 M guanidinium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% N-lauroylsarcosine, 0.1 M 2mercaptoethanol, and passaged through a 23 Ga needle. Lysates were ultracentrifuged through 5.7 M CsCl at 175,000g for 18 h at 22 °C. RNA pellets were washed with 70% ethanol and resuspended in the homogenization solution minus 2-mercaptoethanol. Following three rounds of extraction using 24:1 chloroform:isoamyl alcohol, 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 washed in 80% ethanol, air-dried at room temperature, and stored at 80 °C. Purified RNA was reverse transcribed for RT-PCR using the Superscript III reverse transcriptase (Invitrogen) and the manufacturer’s protocol. All RNA solutions were prepared using diethyl pyrocarbonate-treated water. Following quantification using Quant-IT OliGreen (Invitrogen), 100 ng of cDNA was used to amplify TRP channel sequences.

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continued for 24 h, followed by cryoprotection in 30% sucrose for 12 h at 4 °C, embedding into OCT, and freezing on dry ice. Cryosections (20 lm) were collected on Superfrost plus slides, treated with 1 lg/ml proteinase K for 10 min, and acetylated with 6.61 lM acetic anhydride in 0.1 M triethanolamine for 10 min before overnight hybridization with digoxigenin-labeled RNA probes at 65 °C. Sections were washed with 1  SSC/50% formamide at 65 °C before treating with 20 lg/ml RNAse A at 37 °C, and subsequent washes in 2  SSC and 0.2  SSC applied at 65 °C. Anti-digoxigenin conjugated alkaline phosphatase-labeled sections were developed with 5-bromo-4-chloro-3-indolyl phosphate/4-nitroblue tetrazolium chloride from 3 to 72 h at RT. Sections were extensively washed in 1  PBS (pH 7.4), treated with 4% paraformaldehyde in 1  PBS for 30 min at RT, and coverslipped. 2.5. Buffers The composition of solutions were as follows – low salt buffer: 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 buffer: 5 mM Tris (pH 7.5), 20 mM NaCl, 2% SDS, 2 mM EDTA. All buffers contained protease inhibitors (6 lg/mL aprotinin, 6 lg/mL chymostatin, 1.5 lg/mL leupeptin, 2.0 lg/mL pepstatin A, 0.9 mg/ mL trypsin inhibitor, 4.7 mg/mL benzamide, 1.2 lg/mL e-64, and 0.12 mg/mL pefabloc). 2.6. Preparation and washing of membranes from mouse tissues Tissues were homogenized in low salt buffer on ice and total membranes were collected by centrifugation at 100,000g for 60 min at 4 °C. Membranes were resuspended in high salt buffer by extrusion through an 18 Ga needle and sedimented at 100,000g for 30 min at 4 °C. Membrane pellets were solubilized with membrane buffer and brief sonication on ice followed by centrifugation at 100,000g for 15 min at 4 °C. Protein amounts in solubilized 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 at the ora serrata to remove the cornea; retinas were carefully peeled from RPE/eyecups to limit tissue contamination and immediately frozen on dry ice. Frozen hearts were thoroughly disrupted prior to RNA isolation while frozen retinas and RPE/eyecups were homogenized directly. RNA (20 lg or 10 lg for RPE/eyecup) was resolved using 1% agarose gels containing 0.25 M formaldehyde and transferred overnight to BrightStar positively charged nylon membranes (Ambion). Gene-specific cDNAs were quantified using Quant-IT PicoGreen (Invitrogen) and used to generate high specific activity cDNA probes (>4.0  109 cpm/lg) by random-prime labeling with [a-32P] dCTP (6000 Ci/mmol) using the DECAprime II kit and hybridized at 107 cpm/mL for 15 h at 42 °C in ULTAhyb (Ambion). 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 exposing membranes to phosphorscreens. Phosphorscreens were imaged on a Typhoon image scanner (GE Healthcare). 2.4. In situ hybridization Eyes were carefully dissected from the orbit following lid removal. Extraoccular muscles were severed with scissors to minimize retinal distortion. Following corneal puncture, eyes were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.0) for 1 h at 4 °C. After removal of the cornea and lens, eyecup fixation

Retinas were carefully separated from the eyecup and fixed in 4% paraformaldehyde in Ringer’s buffer (10 mM HEPES (pH 7.4), 130 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 0.02 mM EDTA) for 3 h at 4 °C. Retinas were washed in Ringer’s buffer and embedded in 5% agarose in Ringer’s buffer prior to vibratome sectioning. Sections (150 lm) were pre-blocked for 6 h at 4 °C in 10% donkey serum, 5% BSA, 0.5% fish gelatin, 0.1% Triton X-100 in Ringer’s buffer. Primary antibodies were diluted in blocking solution and applied to tissue sections overnight at 4 °C. After three washes using Ringer’s buffer, Alexa 488 or 555 conjugated antibodies were applied to sections for 1 h at room temperature in blocking solution. Confocal images were acquired using a Zeiss laser scanning microscope LSM 510 (Carl Zeiss Microimaging, Inc.). 3. Results 3.1. Expression of TRP channel mRNAs in the retina 3.1.1. RT-PCR of TRP channel sequences Initially, we used RT-PCR for highly sensitive TRP mRNA detection. PCR primers were designed to amplify across the intron/exon boundaries of published TRP channel sequences (Table S1) (Bult et al., 2008). Amplified sequences were cloned and verified by DNA sequencing (Fig. 1, Table 1). Results revealed that mRNA is present in the retina from all 28 TRP channel genes as well as from the gene

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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 indeed 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

Fig. 1. Detection of transient receptor potential gene transcripts from adult mouse retina cDNA. RT-PCR analysis using 100 ng of cDNA yielded amplicons for all 28 TRP channels as well as PKD1, a TRP-related gene. Multiple splice variants were detected for some TRP channel genes.

for polycystic kidney disease 1 (PKD1), a TRP-associated protein known to bind TRPP2 (Qian et al., 1997; Tsiokas et al., 1997). Multiple splice variants were detected for some TRP channels, including TRPC1, TRPM3, TRPM5, TRPM8, TRPV1, and TRPML1. A second complete set of PCR primers was designed to amplify sequences in the 30 untranslated regions (30 UTR) of TRP channel mRNA (Table S2). RTPCR results using this second set of primers were consistent with results obtained from the first set of primers, confirming that messages from all TRP channel genes are present at readily detectable levels in the retina. Because the retinal pigment epithelium (RPE) is adjacent to photoreceptor outer segments, message from the RPE may be a minor contaminant in RNA of the isolated retina, but the distinct pattern of splice variants observed for some TRP channels in RPE as compared to isolated retina (see Section 3.1.2.), suggests that such contamination is quite low. 3.1.2. Relative abundance of TRP mRNAs by northern blot Northern blots were used to assess the relative abundance of TRP channel mRNA in retina as compared to other tissues (Fig. 2, Figs. S1 and S2, and Table 1). Total RNA was isolated from mouse tissues and probed for TRP channel (and PKD1) messages using the first set cDNAs described above as riboprobes. Transcripts with the strongest retinal signals included TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1 (Fig. 2). Bands for TRPC4, TRPC5, TRPM7, TRPP2, TRPV2, and TRPV4 were clearly visible. Several TRP channel mRNAs were also detected in the eyecup at similar or lower levels as compared to the retina. In contrast, splice variants of TRPM1 and TRPM3 are expressed in the eyecup at concentrations higher than those seen in the retina. In the case of TRPM3, identical transcripts appear to be present in both the retina and eyecup, although with

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 ribosome-rich myoid region (Fig. 3). The relative strength and non-uniformity 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 is likely to be in internal membranes of the photoreceptors and of processes within the OPL. Expression of both TRPM1 and TRPM3 is limited to the inner nuclear layer (INL), but each signal is limited to a different population of cells. Message for TRPM1 occupied a row of neurons adjacent to the OPL, consistent with its known expression in rod bipolar cells (Kim et al., 2008). Antisense probe for TRPM3 hybridized to a population of cells in the middle portion of the INL that appear distinct from rod bipolar cells. ISH using probes prepared from both sets of cDNA sequences failed to reliably detect transcripts for the remaining TRP channels, consistent with northern data suggesting their low abundance in the retina. Based on our results suggesting high expression of TRPM1 and TRPM3 in the eyecup, and the published expression of TRPM1 in melanocytes (Deeds, Cronin, & Duncan, 2000; Fang & Setaluri, 2000), we evaluated the expression of TRPM1 and TRPM3 from albino mice. No significant difference was detected in the level of expression for TRPM1 in albino tissue (Fig. 4), whereas the appearance 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 expression of TRPM1 was detected in the cells of the RPE by ISH, but a strong signal was detected in the ciliary body of the anterior eye segment (Fig. 4B–D) in addition to the expression in the INL. The expression of TRPM1 was further confirmed by immunodetection in both the INL and the ciliary body (Fig. 5). In contrast, TRPM3 exhibits a strong expression in the RPE with a weaker signal in the ciliary body (Fig. 4F–H). 3.3. Immunodetection of TRP channel protein in the retina We tested for protein expression by immunoblots of proteins transferred from SDS PAGE gels of membranes from retina and other tissues, and by immunofluorescence staining of vibratomesectioned agarose-embedded retinas. It is well known that many,

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

RTPCR

Spl. var. amp.

TRPA1 TRPC1

+ ++

2

TRPC2 TRPC3

++++ +++

w ++  brain  heart

TRPC4 TRPC5 TRPC6 TRPC7 TRPM1

+++ +++ ++++ +++ +++++

+  brain + < brain w w ++ < EC  others

TRPM2 TRPM3 TRPM4 TRPM5 TRPM6 TRPM7

++++ +++++ ++++ ++ + ++++

TRPM8 TRPML1

+ ++++

TRPML2 TRPML3 PKD1

++ ++++ +++

TRPP2

+++++

TRPP3 TRPP5 TRPV1 TRPV2

+++ +++ ++++ ++++

TRPV3 TRPV4

++ +++++

TRPV5 TRPV6

+ +

Multiple

2 3

3

3–4

Northerns

ISH

IB

IF

w ++ > brain > kidney

– IS, (ONl, INL, IPL, OPL, some cells in GCL) – Mod. throughout, esp. ONL, INL, OPL ONL, INL, GCL – – – Very strong and specific for INL, ciliary body – INL, RPE (ONL, GCL) – – – –

nt 90 kDa, retina > brain

nt nd

nt 66 kDa; retina < brain; 95 kDa in heart 115 kDa, retina < brain 85 kDa, retina < brain. nt nt Many cross-reactive bands,; does not recognize recombinant protein nt nt nt nt nt 120 kDa, brain > retina

nt Throughout, esp. NFL, GCL, OPL, inner-most INL

– OPL, ONL, (INL, GCL) – – ONL, INL, GCL

nt nt

nt nt

nt nt nt

nt nt nt

INL, GCL (ONL for both sense and antisense) – – – –

Multiple layers; strongest in cone outer segments

– –

Kidney > liver > Retina  brain; bands match recombinant TRPP2 nt nt nt 80 kDa doublet matches recombinant TRPV2; brain > retina Many cross-reactive bands nt

– –

nt nt

nt nt

w ++ < EC  brain w ? w +  EC, brain < kidney ? ++  brain, kidney > EC ? w, EC ++, smear  EC  others +  EC, brain  kidney w, EC w w; diff size m DRG ++  EC, heart < brain w +  EC, brain, heart  kidney w w, EC

Throughout, esp. GCL, INL Inner row of INL nt nt Weak non-specific staining throughout (present in KO) strong in INL (only in WT), soma > dendrites, ciliary body (only in WT) nt nt nt nt nt Cone outer segments

nt nt nt OPL some adjacent to but distinct from ribeye staining; some cells in IPL Müller glia cells 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 peptides from mammalian TRP channels do not give reliable results due to cross-reactivity comparable to or exceeding reactivity with the intended TRP target. We tested many such antibodies, and found most of them unsatisfactory. Results presented here are limited to those that meet one or more of the following criteria (see Table 2 and Table S3): (1) immunoblots indicated a small number of bands, including ones whose mobilities were consistent with the expected protein size (or co-migrating with recombinant protein expressed in yeast), with relative intensities in different tissues qualitatively consistent with Northern results; (2) absence of at least some distinguishable part of the signal in knockout animals (for TRPM1); (3) agreement on specific localization by immunofluorescence with two different antibodies directed against different epitopes; (4) consistency between ISH and immunofluorescence results. A TRPM1 antibody described previously (Morgans et al., 2009) detected TRPM1 (Fig. 5) in the INL and OPL, consistent with expression in ON-bipolar cells. It also recognized TRPM1 in the ciliary body, consistent with results from ISH and northern blots.

TRPC1 (Fig. 6A and B) antibodies reliably detected the b-isoform of hTRPC1 expressed in yeast (80 kDa) as a positive control (Fig. 6B) (Ong et al., 2002), and also detected a major band of the correct size for TRPC1 in both the brain and the retina (93 kDa). Consistent with mRNA levels of TRPC1 detected by northern blotting, TRPC1 protein levels appear higher in the retina than in the brain. The same antibody was not able to detect TRPC1 reliably in retinal neurons by immunofluorescence. The highest levels of immunoreactivity for TRPC5 antibodies were found in membranes of the brain and the retina. A protein with an apparent molecular weight of 97 kDa was detected in both tissues, while a second band 75 kDa was detected only in the brain (Fig. 6D). At longer exposures to enhanced chemiluminescence, a 110 kDa protein, the expected size for full length TRPC5, is also detected in brain and retina membranes. TRPC5 mRNA has been shown to exist as different sized variants (Okada et al., 1998) that may be translated into TRPC5 variants of reduced molecular weight. In addition, a recent publication reported that endogenous TRPC5 from brain lysates migrate lower (97 kDa) than expressed protein (Bezzerides et al., 2004). Taken together,

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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 in the TRPC5 KO mouse (manufacturer’s datasheet), identifies immunoreactive bands with apparent molecular weights other than 110 kDa. Use of this same monoclonal antibody for immu-

nofluorescence detected weak immunoreactivity in all layers of the retina, but a single row of neurons in the INL of the retina showed enhanced TRPC5 immunstaining (Fig. 6G and Fig. S3). This row of neurons, possibly amacrine cells (positive identification

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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 detectable TRPC5 mRNA by ISH, consistent with evidence that TRPC5 mRNA is not abundant within the retina. A TRPM7 antibody detected a protein of 200 kDa (the expected molecular weight for full length TRPM7) in the brain and the retina, as well as protein at 160 kDa that was seen in the brain, retina, and the heart (Fig. 7A). An additional 120 kDa protein was recognized in all tissues tested and is consistent with the

TRPM7 mRNA expression profile. It was previously shown that expression of TRPM7 in cell lines can yield protein at the molecular weights recognized by our antibody (Runnels, Yue, & Clapham, 2001). Lower molecular weight bands may represent cross-reactivity or proteolysis of TRPM7. When used for tissue staining, this antibody detected a specific signal concentrated in the outer segments of cone photoreceptors (Fig. 7D and E). Co-labeling of retinal slices, using two different anti-TRPM7 antibodies, confirmed the

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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 disappeared 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

Fig. 4. Expression of TRPM1 and TRPM3 mRNA in the retina, RPE, and ciliary body of albino mice. (A) Northern blot of TRPM1 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 C57BL/6 albino mouse tissues. Ethidium bromide stained 18S ribosomal RNA control is shown. (B) Localization of TRPM1 mRNA in the albino retina. RPE = retina pigmented epithelium, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Localization of either (C) TRPM1 sense control riboprobe or (D) TRPM1 antisense riboprobe in the ciliary body of albino mice. (E) 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 C57BL/6 albino mouse tissues. Ethidium bromide stained 18S ribosomal RNA control is shown. (F) Localization of TRPM3 mRNA in the albino retina and RPE. Localization of either (G) TRPM3 sense control riboprobe or (H) TRPM3 antisense riboprobe in the ciliary body of albino mice. Scale bars = 50 lm.

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 surprising that mRNA from every TRP channel gene is detectable in the mouse retina. Of course, the technique used, RT-PCR is extremely sensitive, and the presence of a small amount of mRNA does not guarantee that a functionally important amount of protein is present. Moreover, the RT-PCR product intensities cannot be reliably correlated with the levels of message present in the retina. Thus, the results imply that it will be worthwhile to check knockouts of every TRP channel gene for effects on retinal function and health, and considering them as candidate genes for hereditary retinal disease. At the mRNA level, the TRP family members fall into three main groups. One has the highest levels of mRNA as indicated by northern blots, and includes TRPC1, TRPC3, TRPC4, TRPM1, TRPM3, TRPML1, TRPV2, and TRPV4. A second group has weaker but still robust expression, and includes TRPC4, TRPC5, TRPM7, and TRPP2.

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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 of mouse CtBP2 Cytoplasmic N-terminal domain of human TRPC1 Synthetic peptide: 14 amino acids at C-terminus Synthetic peptide: amino acids 827–845 (SKAESSSKRSFMGPSLKKL) of human TRPC5 Synthetic peptide Synthetic peptide Synthetic peptide: amino acids 1817– 1863 at C-terminus of mouse TRPM7 Synthetic peptide: N-terminus of human TRPV2 Synthetic peptide: amino acids 689–968 at C-terminus of human TRPP2

BD Biosciences, 612044

Mouse

0.250 lg/mL (IF)

L. Tsiokas, OUHSC, (Oklahoma City, OK), clone 1F1

Mouse

1.5 lg/mL (IB)

ProSci Inc. (Poway, CA), 3905

Rabbit

2.0 lg/mL (IB), 5.0 lg/mL (IF)

NIH NeuroMab (Davis, CA) 75–104

Mouse

2.0 lg/mL (IB), 5.0 lg/mL (IF)

Sigma (St. Louis, MO), HPA014785 ProSci Inc. (Poway, CA), 46–531 NIH NeuroMab (Davis, CA) 75–114

Rabbit Goat Mouse

2.0 lg/mL (IF) 2.0 lg/mL (IB), 5.0 lg/mL (IF) 5.0 lg/mL (IF)

Santa Cruz Biotechnology (Santa Cruz, CA), SC22520

Goat

1.0 lg/mL (IB), 5.0 lg/mL (IF)

Santa Cruz Biotechnology (Santa Cruz, CA), SC25749

Rabbit

1.0 lg/mL (IB), 2.0 lg/mL (IF)

TRPC1 TRPC3 TRPC5

TRPM1 TRPM7 TRPM7 TRPV2 TRPP2

IF, immunofluorescence; IB, immunoblot.

Expression of the other 16 TRP channel genes was much weaker. The high expression levels motivate pursuing further the potential functions of the 12 most abundant species. However, the less abundant mRNA species could be functionally important. TRPC6 and TRPC7 mRNAs seem to be present at very low levels, but they have been proposed as candidates for the transduction channel of melanopsin-expressing retinal ganglion cells (Sekaran et al., 2007). In addition, a recent publication identified TRPC6 within cells of the INL and the GCL of the retina (Wang et al., 2011). We did not detect any TRPC6 mRNA in the retina by ISH, and northern blotting indicates that TRPC6 mRNA levels are low in the retina. The differences between our study and those of Wang et al. may be due in part to probe differences, but may also result

from differences in protocol for ISH. The high stringency methods we used may have decreased possible signal from low-abundance TRPC6 mRNA. We tested for TRPC6 protein by immunoblotting with two anti-TRPC6 antibodies and they were able to detect TRPC6 protein only in the brain, but not in the retina. At longer exposures, some signal at a size corresponding to TRPC6 signal also appeared in the retina lane, but at those exposures the antibody was cross-reactive with multiple proteins at sizes not expected for TRPC6. Immunostaining of the retina with these antibodies yielded results consistent with the work by Wang et al. Although the antibodies gave weak signal-to-background levels, slightly stronger signal was detected in the lower row of cells in the INL as well as in the GCL.

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Fig. 6. Immunoreactivity of antibodies for TRPC proteins. (A and B), TRPC1 immunoblots from multiple mouse total membrane fractions incubated with secondary HRPconjugated 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 TRPC5immunoreactive 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 reliably detects TRPV1 message in dorsal root ganglion, yet TRPV1 has been reported to play an important role in Ca2+ entry leading to apoptosis in retinal ganglion cells under pressure (Sappington et al., 2009). Sappington et al. used an mRNA probe corresponding to an N-terminal portion of the expressed protein, whereas we used two probes, corresponding to a C-terminal region of the protein as well as the 50 UTR of the TRPV1 mRNA. Both probes failed to show significant signal by ISH of retina cryosections. Paraffin section ISH, used by Sappington et al., has inherent differences compared with our ISH method and may have provided for a more stable signal for low-abundance TRPV1 mRNA. We utilized an anti-TRPV1 antibody and tested it against yeast expressed rTRPV1. While our antibody detected the expressed protein, it failed to detect significant levels of protein in brain or retina, and failed to provide signal by immunostaining of the retina. The antibody we utilized, though specific for TRPV1, may not have been sensitive enough to replicate results from the previous report.

The most striking pattern of localization of both mRNA and immunoreactivity was observed for TRPM1, which is now well established as essential for light responses of ON bipolar cells (Shen et al., 2009). As seen previously (Kim et al., 2008; Koike et al., 2010; Morgans et al., 2009; Nakajima et al., 2009), the staining and mRNA localization in the retina was consistent with expression in these neurons, but most of the staining was in the cell body, not the dendritic tips where mGluR6 is located. A new finding is that TRPM1 is also expressed at even higher levels in the ciliary body, and that the distribution of splice variants there is distinctly different from that in the retina. Complimentary observations were made for the closest relative of TRPM1, TRPM3, which is also expressed strongly in the INL, with weaker mRNA signal in the outer nuclear layer and ganglion cell layer, but very strong expression in the retinal pigmented epithelium. Prior to its implication in ON-bipolar cell signaling, interest in TRPM1 centered on its role in pigmented cells and melanoma (Duncan et al., 1998; Fang & Setaluri, 2000; Miller et al., 2004; Zimov & Yazulla,

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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 antiTRPP2 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 high levels, suggesting a functional role related to pigmentation. A relationship between TRPM3 function and pigmentation is suggested by our observation that albino animals show altered levels of expression of some splice variants as compared to pigmented mice. Decreased expression of shorter TRPM1 variants has been correlated with an increase in metastatic melanomas in human pigmented melanocytes (Duncan et al., 2001; Fang & Setaluri, 2000). Our discovery that variants of TRPM1 are differentially expressed at high levels in either the ciliary body or in the INL of the retina, suggest the interesting possibility that TRPM1 could play a role in some forms of uveal melanoma and/or forms of melanoma-associated retinopathy (MAR) that show selective reduction of b-wave amplitudes in electroretinogram (ERG) waveforms (Keltner, Thirkill, & Yip, 2001; Kim, Alexander, & Fishman, 2008). Another striking expression pattern is that of TRPM7, which two antibodies localize to cone outer segments. TRPM7 has been suggested to be important in homeostasis of Mg2+ or other metal ions,

and it may play such a role in cone outer segments. TRPM7 is a bifunctional channel that could affect cone photoreceptor physiology through its channel activity or its C-terminal alpha kinase activity. Little is known about the endogenous activity and targets of TRPM7 kinase activity, but this activity may be sensitive to changes in the concentrations of intracellular divalent cations (Penner & Fleig, 2007). Because TRPM7 is outwardly rectifying, it exhibits high divalent selectivity with little voltage-dependence over the physiological voltage range for cone photoreceptors. While TRPM7 current is typically small under physiological concentrations of extracellular cations, it is possible that TRPM7 modulates the intracellular concentration of Zn2+, Mg2+, Mn2+, and Co2+ as the intracellular concentration of Ca2+ changes with lightdependent hyperpolarization. It is also possible that TRPM7 may have some function in synaptic vesicle release in cone photoreceptors (Krapivinsky et al., 2006). TRPP2 immunostaining is also enriched in cone outer segments, which are modified cilia, consistent with its previous localization to cilia in the kidney. While TRPP2 is often localized to primary ci-

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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 (Hoffmeister et al., 2011), and was not restricted to the connecting cilia of dissociated outer segments. TRPP2 is also expressed in other neurons throughout the retina, especially in the INL, and may play a role in the cilia of those cells, or in some unknown function. The mRNA for PKD1, a known binding partner for TRPP2, is also detected in all nuclear layers, consistent with their forming a complex as they do in the kidney. Hydrostatic pressure and fluid flow are known to be important for retinal health and it may be that this complex plays a sensing role similar to its role in the kidney. While TRPP2 has not been previously reported in the retina (please note that TRPP2 as used here is identical with the product of the PKD2 gene or polycystin-2, and is not to be confused with TRPP3/PKD2L, which some authors refer to as TRPP2, while referring to TRPP2 as ‘‘TRPP1’’) mutations in TRPP2 may lead to retinal damage (Feng et al., 2009). A recent publication has demonstrated that TRPP2 can interact with retinitis pigmentosa protein RP2 suggesting a possible link between TRPP2 and retinal ciliopathies (Hurd et al., 2010). TRPC1 has been reported to interact with TRPP2 (Bai et al., 2008; Tsiokas et al., 1999), and its expression levels and distribution levels are such that it could easily do so in the retina. However, its levels as indicated by both mRNA and immunostaining appear much higher relative to brain and kidney than do the levels of TRPP2, suggesting that most of the TRPC1 likely has a function independent of that complex. One such function of TRPC1 could include modulating the Ca2+ concentration associated with tonic synaptic activity in photoreceptors (Szikra et al., 2008). TRPC1, TRPC3, TRPC4 and TRPC5 are all expressed robustly in the retina, and, except for TRPC5, are found in multiple cell types, as they are in other tissues, where many functions have been proposed, but not yet unambiguously demonstrated. A common theme may be coupling to activation of PLC-linked GPCR signaling cascades, which are numerous in retinal neurons. The TRPC5 staining of cells whose location suggests they may be amacrine cells, could reflect a function specific for those cells, whose identity will be important to test. TRPC5 is found in hippocampal neurons and has been shown to interact physically

with TRPC1 in the brain (Goel, Sinkins, & Schilling, 2002; Strubing et al., 2001). Though little is known regarding the function of TRPC5 in neurons, the function of TRPC5 in the retina may be analogous to its function in the brain. TRPV2 also has a striking pattern of localization. It has previously been reported that TRPV2 is localized to the plexiform layers of the retina (Leonelli et al., 2009; Yazulla & Studholme, 2004), and our data also demonstrate strong outer plexiform staining using an anti-TRPV2 antibody in 21 day old mice. This staining appears to be localized to the photoreceptor axons, based on its proximity to pre-synaptic ribeye staining. However, we did not detect TRPV2 staining in the IPL as has been reported previously. Both previous studies utilized the same commercial TRPV2 antibody, but neither validated the specificity of the antibody for TRPV2. The antibody we use was confirmed to recognize authentic rTRPV2 expressed in yeast membranes, whose size and identity were further confirmed via an epitope tag expressed at the rTRPV2 terminus. Leonelli et al. (2009) observed sparse immunostaining in the IPL and GCL in the rat retina at P60, but not at P15, so that even if the signal was really due to TRPV2, it may be that P21 mouse retina does not contain detectable levels of TRPV2 in those cell layers. We did not detect TRPV2 immunoreactivity in mouse RGC neurons as was previously reported, but we cannot rule out the possibility that at the age examined the levels of TRPV2 is present in those cells but at levels below our detection limit. In addition to the retina, TRPV2 is localized to the RPE, consistent with our detection of TRPV2 mRNA in the eyecup, and may play a role in thermal regulation of RPE cells (Cordeiro et al., 2010). Although it has been demonstrated that TRPV2 is heat-sensitive (Caterina et al., 1999) additional functional roles have been proposed, and it will be interesting to explore retinal phenotypes of TRPV2 knockout mice. Determining the levels and localization of TRP channel gene expression is only a first step toward elucidating their roles in retinal function, health and disease. However, having the results of such a survey should prove a valuable tool for guiding future work on TRP channel function in the retina as well in less accessible parts of the nervous system.

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