Pannexin Expression in the Cochlea of Mammals:An Alternative Gap Junction

Xiao-Hui Wang, PhD, Michele Streeter, MD, and Hong-Bo Zhao, MD, PhD

Division of Otolaryngology: Head and Neck SurgeryUniversity of Kentucky Chandler Medical Center, Lexington, KY

ABSTRACTOBJECTIVE: To examine the expression of pannexin genes in the mammalian cochlea.STUDY DESIGN: DNA and protein analysis of the mammalian cochlea.SUBJECTS AND METHODS: Cochlear tissues from mice and rats were evaluated for pannexin expression through polymerase chain reaction, Western blots, and immunohistochemistry.RESULTS: RT-PCR shows expression of Panx 1, 2, and 3 in cochlear tissues. Western blots confirm the presence of Panx 1, 2, and 3 in the cochlea. Immunofluorsecent staining localizes Panx 1 at the cochlear lateral wall, the Organ of Corti, and the spiral limbus. Panx2 labeling is visible at the basal cell layer in the stria vascularis. Panx 3 mainly localizes at the cochlear bone.CONCLUSION: Panx 1, 2, and 3 have distinct expression in the cochlea. The function of each gene is likely different. As pannexins form gap junctions, they may provide an alternative to connexins for signal transduction and may be important for hearing.

RESULTSAll three pannexin genes are expressed in the mouse and rat cochleas. Using previously

published primers for both the mouse and the rat brain, we amplified sequence-specific Panx1, Panx2, and Panx3 PCR products [Figure 1]. The previously reported lengths of 279 bp, 269 bp, and 477 bp for mouse Panx 1, 2, and 3 PCR products, respectively were obtained. The lengths of the rat PCR products were 185 bp, 258 bp, and 336, respectively. Mouse Panx3 showed a high level of expression in the cochlea with corresponding low levels of expression in the brain. In contrast, the rat brain showed higher levels of expression of the Panx 2 gene with again strong Panx 3 in the cochlea.

Western blots using either previously published gift antibodies or commercially available antibodies confirm the expression of all three pannexin proteins [Figure 2]. Fig 2A shows the expression of Panx1 in mouse and rat tissues. This corresponds to approximately a 42 kDa protein using a chicken anti-human Panx 1 (4515) antibody. The level of expression of Panx1 appears similar in mouse cochlear, mouse brain, rat cochlear, and rat brain tissue. Figure 2B demonstrates Panx 2 protein expression of approximately 98 kDa. . Figure 2C shows the expression of Panx3, with a monomer around 34 kDa and a dimer at 64 kDa. The monomer dominates in the brain of both rats and mice, whereas the dimer is predominately expressed in the cochlea.

Immunofluorescence using primary antibodies specific for Panx 1, 2, and 3 were used with mouse [Fig. 3] and rat [Fig. 4] cochlear tissue. Similar patterns of expression were found in both mammalian cochleas. Fig. 3A, 3B, and 4A are Panx1 which shows staining along the lateral wall as well as along the basilar membrane with Fig. 3E, 3F, and 4E without the confocal lens. Fig. 3B and 4A reveal more intense staining of Panx1 along the surface of the lateral wall. Fig. 3C and Fig. 4B are Panx2 which shows reactivity along the basilar membrane. Fig. 4C shows staining with Panx2 highlighting the bright expression in the spiral ganglion of the rat. Fig. 3D and Fig. 4 illustrates staining with Panx 3 along the lateral wall, specifically in bone.

REFERENCESBaoBao, L. , L. LocoveiLocovei, S., and Dahl, G. , S., and Dahl, G. PannexinPannexin membrane channels are membrane channels are mechanosensitivemechanosensitive conduits for ATP. conduits for ATP. FEBS FEBS LettLett 572:65572:65--68, 200468, 2004BaranovaBaranova, A., et al. The mammalian , A., et al. The mammalian pannexinpannexin family is homologous to the invertebrate family is homologous to the invertebrate innexininnexin gap junction proteins. gap junction proteins. GenomicsGenomics 83:70683:706--716, 2004.716, 2004.BarbeBarbe, M., , M., MonyerMonyer, H., and , H., and BruzzoneBruzzone, R. Cell, R. Cell--Cell Communication Beyond Cell Communication Beyond ConnexinsConnexins: The : The PannexinPannexin Channels. Channels. PhysiologyPhysiology 21:10321:103--114, 2006.114, 2006.BruzzoneBruzzone, R., et al. , R., et al. PannexinsPannexins, a family of gap junction proteins expressed in brain. , a family of gap junction proteins expressed in brain. Proc Proc NatlNatl AcadAcad SciSci USAUSA 100:13644100:13644--13649, 2003.13649, 2003.Guilford P., et al.Guilford P., et al. A nonA non--syndrome form of syndrome form of neurosensoryneurosensory, recessive deafness maps to the , recessive deafness maps to the pericentromericpericentromeric region of chromosome 13q. region of chromosome 13q. Nat GenetNat Genet 1994;1994; 6:24.6:24.KelsellKelsell D., et al.D., et al. ConnexinConnexin 26 mutations in hereditary non26 mutations in hereditary non--syndromicsyndromic sensorineural deafness. sensorineural deafness. NatureNature 1997;1997; 387:80.387:80.PanchinPanchin, Y. et al. A ubiquitous family of putative gap junction molecu, Y. et al. A ubiquitous family of putative gap junction molecules. les. CurrCurr BioBio 10:47310:473--474, 2000.474, 2000.PanchinPanchin, Y. Evolution of gap junction proteins , Y. Evolution of gap junction proteins –– the the pannexinpannexin alternative. alternative. JournJourn Exp Bio.Exp Bio. 208:1415208:1415--1419, 2005.1419, 2005.StebbingsStebbings, L., et al. Gap junctions in , L., et al. Gap junctions in DrosophilaDrosophila: developmental expression of the entire : developmental expression of the entire innexininnexin gene family. gene family. MechMech DevDev 113:197113:197--205, 2002.205, 2002.Vogt, A., et al. Pannexin1 and Pannexin2 expression in the deveVogt, A., et al. Pannexin1 and Pannexin2 expression in the developing and mature rat brain. loping and mature rat brain. MolecMolec Brain Brain ResRes, 2005., 2005.

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METHODS AND MATERIALSAs previously reported (Yu et al, 2006), animals were anesthetized with pentobarbital and decapitated. The temporal bone was then removed, and the cochlea isolated. Analysis of PanX1 and PanX2 expression (RT-PCR): Purifed total RNA (RNeasy Kit, Qiagen, Valencia, CA) was extracted from mouse and rat cochleas and hippocampus. Primers for rat brain Px1 and Px2 as described in Vogt, 2005 were used. Reverse transcription was performed using the iScript one-step RT-PCR kit with SYBRGreen (Bio-Rad, Hercules, CA). To detect pannexin cDNAs, PCR was performed by adding 1 μl of the forward and reverse primers, 10 μl of the 2X SYBR mix, and 0.4 μl of iScript reverse transcriptase. DNA amplification conditions included an initial 5-minute denaturation step at 95ºC and 35 cycles of 30 s at 95ºC, 30 s at 60ºC, 40 s at 72ºC, and finally, 5 minutes at 72ºC.Western Blot :Cochleas were collected in CelLytic MT Tissue Lysis/Extraction Reagent (C3228 Sigma-Aldrich, St. Louis MO) supplemented with phenylmethylsulfonyl fluoride (PMSF 100 µg/ml), a protease inhibitor cocktail (1:50, P8340, Sigma-Aldrich, St. Louis MO), thimerosal (1:10,000) and DNase (10µg/ml). After homogenization with a Kontes pellet pestle, lysates were centrifuged at 3,000 g for 15 min at 4°C. The cochlear sample was then diluted in an equal volume of 2X loading buffer (lithium dodecyl sulfate (LDS) Laemmli buffer with 100 mM dithiothreitol (DTT), heated to 100° C for 5 min or not heated, and spun 10 min before loading onto a 7.5% LDS–acrylamidegel. After electrophoresis, the gel proteins were electrotransferred onto a nitrocellulose membrane, which was blocked with blocking solution (2% non-fat dry milk/2% bovine serum albumin in PBS) and then incubated with the goat anti-human pannexin-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit anti-mouse pannexin-2 antibody (Invitrogen, Carlsbad, CA), also in blocking solution. After washing with Tris-buffered saline containing 0.05% Tween 20 (TBST), the membrane was incubated with an HRP-conjugated secondary antibody (1:10,000, donkey anti-goat IgG or donkey anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove PA) in blocking solution containing 1% normal donkey serum. Finally, after thoroughly washing with TBST, prestin expression was detected using an enhanced chemiluminescence Western blotting detection system (Amersham Biosciences, Piscataway NJ). The apparent molecular masses were calculated by non-linear curve fitting of the molecular mass standards and indicated on each gel. After development, the film was photographed and analyzed using Kodak ID Image Analysis Software.Comment: 1) For cells extraction using either CelLytic M Lysis/extraction reagent (Sigma C2978), or homogenize with dounce tissue grinder (Wheaton) in 10 mM Tris buffer, pH 7.5 supplemented with 50 mM sucrose, 1mM EDTA and protease inhibitor cocktail. After centrifuged to get rid off the nucleus, the supernatant was mixed with 2 X LDS Laemmli loading buffer without boiling.2) We use lithium dodecyl sulfate (LDS) instead of SDS to avoid aggregation. Immunohistochemistry: The otic capsule was dissected in the standard extracellular solution (142 NaCl, 5.37 KCl, 1.47 MgCl2, 2 CaCl2, 10 HEPES in mM, 300 mOsm, pH 7.2) to expose the organ of Corti. After the tectorial membrane and stria vascularis were removed, the sensory epithelium was picked away with a sharpened needle and collected for staining. The isolated senosory epithelium was further incubated in standard extracellular solution with trypsin (1mg/ml) and shaken for 5-15 minutes. The dissociated cells were transferred to a dish for staining and fixed with 4% paraformaldehyde for 30 minutes. After three washes with 0.1 M PBS, the dissociated cells were incubated in blocking solution (10% goat serum and 1% BSA in PBS) with 0.1% Triton X-100 for 20 minutes. The epithelia or cells were incubated with polycolonal goat anti-human Pannexin-1 or polyclonal rabbit anit-mouse Pannexin-2 at a dilution of 1:400 (sources same as for Western blotting) in the blocking solution at 4ºC overnight. After being washed in 0.1 M PBS three times, the epithelia and cells were reacted to a mixture of the secondary antibodies, which were Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG (1:500; catalog No. A11029 and A11036, respectively; Molecular Probesm, Eugene, OR), in the blocking solution at room temperature for 1 hour. After completely washing out the second antibodies with 0.1 M PBS, staining was observed under a confocallaser scanning microscope. In some cases, the cell nuclei were visualized by staining with 4,6-diamidino-2-phenylindole (DAPI, D1306; Molecular Probes). The stock solution of DAPI (5 mg/ml) was made with deionized water. After the reaction to the seconday antibodies, the epithelia or cells were incubated with a 1:50 dilution of DAPI stock solution for 15 minutes at room temperature (23°C).The stained epithelia or cells were observed under a Leica confocal microscope (Leica TCS SP2) equipped with a ×40 (N/A 1.25) or a ×100 (N/A 1.4) apochromatic oil objective. The argon (488 nm) laser and krypton (568 nm) lasers with 500-530 nm and 600-665 nm emission filters were used for visualization of Alexa Fluor 488 and 568, respectively. DAPI staining was observed under a multiphoton laser with a 388-478 nm emission filter. Machine cross-talking was checked by the standard fluorescent slides and turning on/off of one laser in dual-laser excitation. All images were saved in the TIFF format. Images were assembled in Photoshop (Adobe Systems, Mountain View, CA) for presentation. For quantitative analysis of pannexin expression in the cochlear sensory epithelium, serial sections of the confocal image were taken from the apical surface to the basal bottom of the epithelium along the Z-axis. The pixels of Panx1 and Panx2 labeling in 100-m long epithelium were calculated in each section and then summed. The staining intensities of PanX1 and PanX2 were also measured by use of the plot profile function in ImageJ (NIH, Bethesda, MD; Yu et al., [2006]). Data were plotted in SigmaPlot (SPSS Inc., Chicago, IL).

INTRODUCTIONAn estimated 70% of hereditary hearing loss is nonsyndromic. The first autosomal-

recessive nonsyndromic deafness (AR NSD) gene, GJB2, encodes a protein called connexin26 (Cx26) (Kelsell, 1997). This protein oligermizes with five other similar proteins to form a connexon. Two connexons that dock together from neighboring cells form a gap junction. The Cx26 gene is thought to be essential for potassium recycling. This gene is expressed in the stria vascularis, nonsensory epithelial cells, the spiral ligament, and the spiral limbus.

While connexins are found in vertebrates, an unrelated family of proteins called innexinsare found in invertebrates. Innexins are not functionally equivalent to connexins, but engage in similar roles in synaptic transmission, embryonic and postembryonic development, and morphogenesis (Stebbings, 2002). Using PCR with degenerate primers, Panchin et al.(Panchin, 2000) found two putative innexin-like sequences in the human genome, collectively called pannexins. These genes are gap-junction proteins (Panx1 and Panx2). Since that time, a third protein called pannexin 3 has been discovered.

Despite the lack of similar sequences with connexins, pannexins have strong similarities at the structural and functional level. Thus far, there have been no studies that have attempted to localize the expression of Panx1, Panx2, or Panx3 to the inner ear. We aim to prove the presence of pannexin expression in the cochleas of mammals, thus providing an alternative to connexins for signal transduction through gap junction proteins.

FIGURESFig 3. Mouse Fig 3. Mouse PanxPanx 1, 2, and 3 1, 2, and 3 immunofluorescenceimmunofluorescence Fig 4. Rat Fig 4. Rat PanxPanx 1, 2, and 3 1, 2, and 3 immunofluorescenceimmunofluorescencein cochlear tissue. A/E is Panx1 along the lateral in cochlin cochlear tissue. A/E is Panx1 along the lateral in cochlear tissue. A/E is Panx1 along the lateralear tissue. A/E is Panx1 along the lateralwall and basilar membrane. B/F is a higher power wall. B/F iswall and basilar membrane. B/F is a higher power wall. B/F is Panx2 along the basilar membrane.Panx2 along the basilar membrane.Panx1. C/G is Panx2 along the basilar membrane, C/G is Panx2 Panx1. C/G is Panx2 along the basilar membrane, C/G is Panx2 in the spiral ganglion. D/H is in the spiral ganglion. D/H is D/H Panx3 along the lateral wall (especially bone). Panx3 alonD/H Panx3 along the lateral wall (especially bone). Panx3 along the lateral wall (especially bone).g the lateral wall (especially bone).

FIGURESFig 1. Fig 1. PannexinPannexin 1, 2, and 3 PCR products. Fig 2. 1, 2, and 3 PCR products. Fig 2. PannexinPannexin 1, 2, and 3 Western Blot1, 2, and 3 Western BlotPanxPanx--specific primers for mouse and rat were used. Fig. 2A showspecific primers for mouse and rat were used. Fig. 2A shows s PanxPanx 1 proteins, corresponding1 proteins, correspondingThe expected lengths of 279 The expected lengths of 279 bpbp, 269 , 269 bpbp, and 477 , and 477 bpbp to a 42 to a 42 kDakDa protein. Fig. 2B shows the higher protein. Fig. 2B shows the higher for mouse for mouse PanxPanx 1, 2, and 3 PCR products, respectively expression of Panx2 in 1, 2, and 3 PCR products, respectively expression of Panx2 in the cochlea vs. brain.the cochlea vs. brain.were obtained. The lengths of the rat PCR products Fig. 2Cwere obtained. The lengths of the rat PCR products Fig. 2C is Panx3, with a monomer of 34 is Panx3, with a monomer of 34 kDakDawere 185 were 185 bpbp, 258 , 258 bpbp, and 336 , and 336 bpbp, respectively. and a , respectively. and a dimerdimer of 64 of 64 kDakDa. . Mouse cochleaMouse cochlea Mouse brainMouse brain Rat cochlea Rat brainRat cochlea Rat brain(Lane 2(Lane 2--4) 4) (Lane 5(Lane 5--7) (Lane 97) (Lane 9--11) (Lane 1211) (Lane 12--14)14)

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Mouse Cochlea Mouse Brain Rat Cochlea Rat BrainB.

Rat Cochlea Mouse Cochlea Rat Brain Mouse BrainC.

Mouse Cochlea Mouse Brain Rat Cochlea Rat Brain

CONCLUSIONPanx 1, 2, and 3 have distinct expression in the cochlea. The function of each gene is

likely different. As pannexins form gap junctions, they may provide an alternative to connexins for signal transduction and may be important for hearing.

DISCUSSIONConnexons form gap junctions, which provide for rapid signal transduction between

cells. Their function is essential for hearing, as exemplified by the mutations in the connexin gene GJB2 gene, which have been found to be the cause of a significant proportion of nonsyndromic deafness worldwide. Pannexins are an unrelated family of gap junction proteins that have recently been discovered in the mammalian genome. To date, there have been no reports on the expression of the pannexin genes in the cochleas of mammalian species. Our research, through PCR, Western blots, and immunofluorescence, has proven the presence and expression of Panx1, Panx2, and Panx3 in both mouse and rat cochleas. The function of each gene is likely different as they localize in distant portions of the cochlea and have different levels of expression.

At this time, future directions of research should include the analysis of the physiologic function of the pannexin proteins as well as the expression of pannexins during development.

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