Molecular Vision 2006; 12:1417-1426 <http://www.molvis.org/molvis/v12/a160/> Received 21 September 2006 | Accepted 14 November 2006 | Published 17 November 2006

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Molecular characterization of pannexins in the lens

Galina Dvoriantchikova,¹ Dmitry Ivanov,^1,2 Anna Pestova,^1,2 Valery Shestopalov^1,3
 
 

¹Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, FL; ²Vavilov Institute of General Genetics RAS, Moscow, Russia; ³Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL

Correspondence to: Valery Shestopalov, Ph.D., Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Miami, FL, 33136; Phone: (305) 547-3680; FAX: (305) 547-3658; email: vshestopalov@med.miami.edu

Abstract

Purpose: Cell communication in the lens is critical for the life-long homeostasis of this tissue. Abundant gap junctions and cell-cell fusions are reported to be indispensable to the metabolic requirements and optical properties of the highly interconnected syncytial lens tissue. The expression of the recently characterized Panx1 and Panx2 gap junction proteins in the lens is, therefore, rather intriguing. Co-expression of pannexins and abundant connexins in the lens suggests that the two gap junction protein families have distinct roles in cell communication.

Methods: Panx1 and Panx2 expression was studied by in situ hybridization and quantitative RT-PCR. We examined properties and tissue distribution of Panx1 isoforms by Western blot analysis. Immunohistochemistry was used to visualize lens regions that accumulate Panx1 to study intercellular localization and spatial relationship with lens connexin gap junctions.

Results: Panx1 and Panx2 expression peaked in lens epithelial cells prior to differentiation. We detected one ubiquitously expressed Panx1 isoform and two additional isoforms that were only detected in the lens and the retina. Our results indicated that the ubiquitous 58 kDa and the oligomeric 120 kDa isoforms were plasma membrane-bound, resistant to Triton X-100 treatment, and was likely associated with cholesterol-enriched membrane microdomains. Immunohistochemistry revealed Panx1-specific punctuate labeling in the plasma membrane, and intensive labeling of the organelles in the epithelial and immature fiber cells. In addition, we detected Panx1 immunoreactivity in blood endothelial cells of the tunica vasculosa lentis capillaries and in blood erythrocytes.

Conclusions: Despite similarity in detergent solubility of pannexins and connexins, the lack of spatial co-localization in the lens membranes suggested a distinct, non-redundant to connexin function for these proteins in the membrane.

Introduction

Gap junctions are composed of proteins that form a channel piercing membrane that allows passage of ions and small molecules, and thus mediating one of the most common forms of cell-cell and cell-media communication [1]. Gap junctions are present in various tissues and are highly abundant in the lens of the eye [2-4]. The ocular lens is an avascular tissue that relies extensively on the gap junction-mediated communication between fibers to support metabolic requirements [3,4]. The importance of gap junctions for stable lens function was confirmed in experiments with targeted knockout of genes for lens connexins, which resulted in microphthalmia and cataract formation [5,6]. Significantly, connexin-based hemichannels that were also found in the lens represent a pathway for ionic balance and volume regulation in this tissue [7].

Until recently, connexins were suggested to be the sole conduits for the electrical coupling found across the vertebrate body [8]. This situation changed when a new family of gap junction molecules, pannexins, was identified in various taxonomic groups of vertebrates [9-12]. The exogeusly expressed rodent Panx1 alone and in combination with Panx2 was demonstrated to induce formation of intercellular channels in paired oocytes [9] and shown to allow passage of Ca²⁺ between contacting human prostate cancer cells [13]. Although Panx2 does not form homotypic channels, it might participate in heterotypic channel formation, providing a modulatory effect on Panx1 channels [9]. Significantly, Panx2 expression has been reported to be predominantly expressed in the central nervous system (CNS), thus suggesting that the modulation of heteromeric channel properties is required for neuron-specific functions [9,14,15]. When expressed in single Xenopus oocytes, Panx1 hemichannels were shown to be functional in plasma membranes [9,16,17]. This type of nonjunctional function has been previously reported for connexins [17,18]. However, it remains unclear whether lens pannexins junctionally duplicate connexins or play a distinct physiological role.

Panx1, the most studied member of the family, forms hemichannels with unique physiological properties with the ability to support large currents during channel opening and with permeability to Ca²⁺ and ATP [13,17,19]. While voltage and pH gating properties of Panx1 are similar to those of connexins, the permeability profile of the Panx1 channels and their insensitivity to Ca²⁺ distinguish them from the connexin-based ones [9,17]. Recent analysis of the Panx1 hemichannel properties demonstrated their mechanosensitivity - the ability to open at physiological concentrations of Ca²⁺ and to allow the passage of signaling molecules such as ATP [17,19].

A putative role in cell communication suggests that pannexins might be involved in lens homeostasis, maintenance, and metabolic integration to complement the connexins function. However, data from earlier experiments with connexins knockout mice that completely disrupted electrical coupling in the lens [5,20,21], lends weight to the argument for a distinct function of pannexins. In this work, we have characterized Panx1 and Panx2 expression, the protein isoforms and cellular localization of Panx1 in murine lens and in blood endothelial cells of tunica vasculosa lentis, which provide the blood supply to neonatal rodent lenses. Our results indicate divergent, rather than redundant, functions of the two families of lens junction proteins.

Methods

Animals and cell lines

All experiments were performed in compliance with the ARVO statement for use of animals in ophthalmic and vision research according to the IACUC approved protocols. C57BL/6 mice were housed in animal care facilities according to NIH guidelines (NIH publication number 86-23, 1985) and University of Miami IACUC approved protocols. Neonatal P2-P5 lenses were used for immunohistochemistry; juvenile P20 lenses were utilized to microdissect differentiation stage-specific cells for protein analysis. Adult animals were euthanized by CO₂ inhalation and neonates by cervical dislocation, according to the IACUC approved protocol.

HeLa S3 cells (ATCC number CCL-2.2) were cultured at 37 °C with 5% CO₂ in DMEM media supplemented with 10% FBS. Endogenous expression of Panx1 in these cells were confirmed by three independent RT-PCR experiments using the gene-specific primers for Panx1 (5'-caa ggg aga gga cca ggg c-3'; 5'-atc tat tct tct atg acg ctg-3') and DNAse-treated total RNA preparation.

Quantitative RT-PCR

For the purposes of real-time RT-PCR analysis, whole lenses were dissected from mouse eyes in sterile RNase-free PBS. Lenses were microdissected into three regions for RNA extraction: lens epithelium, young elongating fiber cells and mature fiber cells. Total RNA from these regions was isolated using Absolutely RNA® microprep kit (Stratagene, La Jolla, CA) according to manufacturer's instructions. After reverse transcription (Message Sensor^TM RT Kit), the cDNA was PCR-amplified using gene-specific primers for Panx1 (5'-caa ggg aga gga cca ggg c-3'; 5'-atc tat tct tct atg acg ctg-3'), Panx2 (5'-gag aaa aag cat acc cgc cac-3'; 5'-ggg tga gca gac atg gaa tg-3'), Cx50 (5'-gca aga gag aaa gac agc ac-3'; 5'-cca gag gcg gat gtg tga g-3'), and Actb (5'-cac cct gtg ctg ctc acc-3'; 5'-gca cga ttt ccc tct cag-3'). We used QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA) and Bio-Rad I-Cycler real-time PCR machine. Primers were designed to be spaced with an intron to avoid amplification from DNA templates; all primers were synthesized and HPLC-purified by Sigma. The amplification products were sequenced at one strand to verify the product specificity in preliminary experiments. The PCR conditions were as follows: 95 °C, 15 min; 40 cycles beginning at 94 °C for 30 s; 58 °C for 30 s; and 72 °C for 1 min. For Panx1, Panx2, Cx50, and Actb, transcript quantification was measured with the I-Cycler simultaneously in each separate experiment (n=3) and relative transcript abundances calculated. We used a relative quantification approach to compare transcript abundances and followed a method described in reference [22]. Relative quantification determines the changes in steady-state mRNA levels of a gene across multiple samples and expresses it relative to the levels of an internal control RNA, the Actb (β-actin) gene in our experiments. The sizes of the amplified PCR products were confirmed by gel electrophoresis after each I-Cycler experiment. An unpaired t-test was used for statistical analysis (Graph Pad Software, Inc., San Diego, CA). A p<0.05 was considered statistically significant.

In situ hybridization

For in situ hybridization, P5 lenses from C57BL/6 mice were fixed in 4% paraformaldehyde and sectioned to 5 μm thickness. Plasmid DNA was purified from cDNA clones (ATCC clone number 7067302 for Panx1 and Invitrogen cDNA clone Image:4501852 for Panx2). To verify the identity of mouse Image, cloned cDNA was completely sequenced and submitted to GenBank (DQ093579). The endonuclease-digested plasmid DNA was transcribed into dioxigenin-labeled sense probe control RNA probes and antisense probes using a DIG RNA labeling Kit (SP6/T7) using the procedure provided by the manufacturer (Roche Diagnostics, Castle Hill, NSW, Australia). In situ hybridization using a DIG-labeled cRNA probe was performed according to the protocol provided by the manufacturer (Boehringer Mannheim, Indianapolis, IN) as described [17]. For each in situ experiment, signals for antisense and sense (control) probes were compared between neighboring sections of the same mouse lens, which were processed simultaneously.

Antibodies

The following commercially available antibodies were used: mouse monoclonal anti-Cx50 (Zymed); mouse monoclonal CDF4 antihuman golgin-97 (Molecular Probes/Invitrogen, Carlsbad, CA), mouse monoclonal anti-Protein Disulfide Isomerase (QED Bioscience Inc, San Diego, CA). The rabbit polyclonal anti-Panx1 antibody was raised against the truncated 18 kDa carboxyl terminus of human PANX1 protein; the corresponding cDNA was derived from the clone IMAGE: 4390851 as described in reference [23]. After affinity purification, these antibodies were used for western blot (dilution 1:3,000) and immunohistochemistry (dilution 1:1,000).

Western blot analysis

To analyze membrane proteins, we pooled 10-12 freshly microdissected P20 mouse lenses, homogenized them without detergents, and fractionated them by centrifugation to obtain fractions enriched with integral membrane proteins, organelle membranes, and water soluble proteins. Lenses were rinsed in detergent- and Ca ²⁺-free TEE buffer containing 5 mM Tris pH 7.5, 5 mM EDTA, 5 mM EGTA, 0.25 M sucrose. After homogenization with a Teflon pestel, a brief 30 s sonication, and centrifugation at 500x g for two min to removal cell debris, total lens homogenates were spun at 7,000x g for 30 min at 4 °C. The supernatant was subsequently spun at 100,000x g for 1 h at 4 °C to separate the microsomal fraction enriched in organelles membranes from water-soluble cytosolic proteins. Fractionated samples were solubilized in the sample buffer (Invitrogen, Carlsbad, CA), adjusted for the total protein, separated by SDS PAGE using 4-12% gradient gels (Invitrogen) in denaturating conditions, transferred to PVDF membranes (Invitrogen), and probed with antibodies. To analyze Panx1 in blood endothelial cells, the tunica vasculosa lentis envelopes from neonatal (P3) lenses were harvested by stripping away from neonatal lenses under the dissecting microscope, pooled, and homogenized following a process similar to that used on lens fiber cells. Erythrocyte membrane ghosts were prepared by hypotonic lysis of citrated murine whole blood using a modification of a method by Galo et al. [24]. Briefly, pelletted red blood cells were mixed with lysis buffer (20 mM Tris, pH 7.4) at a volumetric ratio of 100:1 (buffer to cells) for 60 min, and then isolated by centrifugation at 20,000x g for 20 min at 2-8 °C. Purified erythrocyte ghosts and blood endothelial cells were solubilized in the sample buffer, equilibrated for the total protein and analyzed by western blot as per the afore described method.

To detect differentiation-regulated changes in Panx1 accumulation, we microdissected P7 lenses into three regions using a protocol described in reference [25]. Briefly, thick (500 nm) unfixed lens slices were microdissected into (1) lens epithelial cells (EC) by peeling away capsule with attached cells; (2) young elongating (cortical) fibers (YF) and (3) mature, fully elongated fibers (MF) connected to sutures, using fine tip forceps under the dissecting microscope. Dissected cells from corresponding lens regions were pooled together. To assess the relative abundance of Panx1 and Cx50 proteins in central and superficial lens regions characterized by different crystallin content, we analyzed membrane-enriched water-insoluble fractions extracted from pooled samples adjusted for total protein. To detect Panx1 isoforms that are tolerant to Triton X-100 treatment, we stripped pelleted membranes by rinsing them in buffer containing 20 mM NaOH and re-suspending them in TEE containing either 1% Triton X-100 (TX-100), 10 mM methyl-beta-cyclodextrin (MCD, Sigma) or both for 60 min on ice. To separate insoluble proteins, resistant to the detergent, from solubilized ones, we spun extracts at 18,000x g. Both the pelleted membrane proteins and supernatants were analyzed by western blot. Membrane-enriched fractions were first probed with primary antibodies against Panx1 then stripped and reprobed with antibodies against Cx50. Band detection was performed using secondary HRP-conjugated antibodies and chemiluminescent Super Signal substrate (Pierce Biotechnology, Rockford, IL).

Immunohistochemistry and microscopy

Lenses from C57BL/6 mice were fixed in 4% paraformaldehyde, sectioned to a thickness of 100 μm with a vibratory microtome (Vibratome, St. Louis, MO) and immunostained using a protocol described in reference [26]. Distribution of primary antibodies was visualized by staining with AlexaFluor488 and/or Fluor 546 dye-conjugated secondary antibody (Invitrogen/Molecular Probes). Control sections were incubated with nonimmune rabbit serum instead of primary antibodies. Imaging for the specific fluorescence labeling in thick lens slices was performed using Leica TSL AOBS SP5 confocal microscope (Leica Microsystems, Exton, PA). To visualize polymerized actin, we incubated fixed and permeabilized (Triton X-100 0.2% in PBS, 1 h) lens slices with phalloidin-Alexa 647 (Invitrogen/Molecular Probes, 1:300 dilution in PBS). DAPI (1:10,000 dilution in PBS, Molecular Probes) was used to label DNA in the nuclei.

Results

Pannexins expression in the lens

We analyzed the expression patterns of Panx1 and Panx2 genes in the mouse lens using regular and quantitative RT-PCR for the mRNA extracted from the regions with distinct differentiation status (Ep, YF and MF) and compared the expression to that of connexin50/a3 (Cx50) gene encoding the gap junction protein abundantly expressed in the lens. Alternatively, Panx1and Panx2 transcript accumulation has been studied using in situ hybridization in the paraffin sections of P5 mouse lenses. Our regular RT-PCR data indicated that both pannexins were expressed in all three lens regions (Figure 1). After normalization to the level of Actb mRNA, quantitative RT-PCR data showed that Cx50 transcripts were considerably more abundant than transcripts for Panx1 and Panx2 in the regions of the lens that we studied. When the ratio of Panx1, Panx2 relative to Cx50 was calculated, the mRNAs for both pannexins appeared to be significantly less abundant as compared to Cx50: approximately ten fold in the lens epithelium and more than a hundred fold in mature fiber cells (Figure 2). P-values for gene expression levels, calculated using an unpaired t-test, varied in the following ranges: 0.0001-0.001 for Panx1; 0.0003-0.002 for Panx2; 0.001-0.007 for Cx50. In contrast, the level of Cx50 mRNA decreased only three fold in elongated fiber cells as compared to that in the lens epithelium. In good agreement with the qPCR data, in situ hybridization showed Panx1 and Panx2 expression in neonatal lenses co-localized to the lens epithelium and YF (Figure 3).

Panx1 protein accumulation in the lens

Panx1 accumulation in lens cells has been examined by Western blot and immunohistochemistry with anti-Panx1 polyclonal antibodies. In the total lens protein extract, we detected two major bands (43 and 120 kDa) and two minor bands (58 kDa and 62 kDa; Figure 4). Pannexins are integral membrane proteins with four predicted membrane spanning domains. We, therefore, examined the association of Panx1 with cell membranes. First, to identify the potential membrane-bound isoforms, we fractionated whole lens homogenates prepared without detergents by centrifugation. We analyzed the water-soluble and water-insoluble fractions, enriched with membrane proteins, by Western blot. Low speed (7,000 g) centrifugation of whole lens homogenates quantitatively separated the 43 kDa isoform, which remained in the supernatant, from the 58 kDa and 120 kDa isoforms, that pelleted together with the plasma membrane-enriched fraction (Figure 4, lane 3). The 62 kDa protein was subsequently pelleted along with the microsomal fraction after further spinning the supernatant at 100,000 g (Figure 4, lane 4). Second, we tested the membrane-bound Panx1 (all but 43 kDa isoform) for resistance to extraction by 1% Triton X-100 and methyl-β-cyclodextrin (MCD). In contrast to membrane-associated peripheral proteins, many integral membrane proteins, like connexins, form aggregates in lipid rafts membrane microdomains, which cause them to become resistant to extraction with 1% Triton X-100 [27,28]. Such aggregates, however, render solubility to Triton X-100 after the lipid rafts are disrupted with the cholesterol-depleting drug MCD [29]. Our data indicated that the 58 kDa and 120 kDa Panx1 isoforms were indeed resistant to Triton X-100, with no immunoreactivity detected in the supernatant after treatment and centrifugation for 20 min at 18,000x g (Figure 5; upper panel, lanes 2 and 3). Treatment with MCD alone also failed to extract Panx1 from the membranes (lanes 4, 5). However, a combined Triton X100-MCD treatment extracted a significant portion of both 58 kDa and 120 kDa Panx1 from the lens membranes (lane 6) into the supernatant recovered by a 18,000x g spin (lane 7). Re-probing the same blots with anti-Cx50 antibodies revealed a close match between the solubility of the membrane-bound isoforms of Panx1 and Cx50. Both proteins could only be extracted from the membranes by a combined treatment with TritonX-100-MCD (Figure 5, lanes 6 and 7). The presence of 62 kDa isoform in the water insoluble pellet prior to extraction in this experiment (lanes 1 and 2 on Figure 5) can be explained by insufficient sonication of the larger sample in this preparation.

We then compared Panx1 abundance in plasma membranes of cells from different tissues with that of the lens by Western blot. In non-CNS tissues, the 58 kDa Panx1 was the most abundant, while the other isoforms were present at a low level. Accumulation of significant amounts of the 120 kDa oligomer and the 60/62 kDa isoforms (Figure 6, asterisks) were, therefore, specific to the retina and lens. Next, to detect changes in Panx1 accumulation during development, we also examined water-insoluble proteins extracted from the three lens regions distinct in differentiation status. In the microdissected lens samples, we found the highest accumulation of the total membrane-bound Panx1 protein in the YF and MF lens regions, with significantly lower levels in the EC region (Figure 6, lanes 6-8). Lens epithelial cells expressed predominantly the 58 kDa isoform and showed the lowest amounts of the Panx1 oligomer (Figure 6, lane 6). In the YF and MF regions, Panx1 was detected primarily in the oligomeric form. A significant decrease in the 58 kDa isoform and an increase in the 62 and the 120 kDa isoforms occurred in the MF as fiber cell maturation progressed to a completion. The 62 kDa isoform was not detected in cells microdissected from the EC region (Figure 6, lanes 4, 6, 7).

Panx1 immunohistochemistry

Our immunostaining data with polyclonal anti-Panx1 antibodies showed that the protein was predominantly concentrated in the lens cortex and that the concentration decreased toward the lens nucleus (Figure 7). High resolution confocal microscopy revealed Panx1-specific labeling in both plasma and intracellular membranes of the perinuclear region of lens epithelial and fiber cells. Panx1-specific labeling partially co-localized with endoplasmic reticulum (ER) and Golgi in the double labeling experiments of HeLa cells that express Panx1 endogenously (Figure 8). HeLa cells were used because a similar experiment was not possible in lens slices, where anti-Golgin antibodies failed to work. The asymmetrical distribution of Panx1, observed in the perinuclear region in HeLa cells, was similar to the pattern observed in peripheral fibers (Figure 7C). Endogenous expression of Panx1 in HeLa cells was detected in three independent experiments and was confirmed by the results of microarray studies obtained in other laboratories working with this popular cell line (GEO database accession GDS477, GDS705, GDS885, GDS478, GDS1212, and GDS449). We found that in the lens this pattern persisted until the denucleation process became apparent in the MF of the lens cortical region. As the differentiation status of the lens cells altered, Panx1 localization changed from being predominantly cytoplasmic in epithelial and YF, to becoming increasingly associated with the plasma membrane in fibers from the YF and MF regions (Figure 7B,C). In the deeper cortex, where nuclear picnosis characteristic of denucleation became apparent, Panx1 labeling of the fiber cell membranes considerably diminished (Figure 7D). Endothelial cells of the tunica vasculosa lentis that remained attached to the lens capsule in our preparations also exhibited Panx1-specific labeling of the plasma membrane and the perinuclear region (Figure 7E,F). It is to be noted that specific labeling of Panx1 was localized predominantly to the luminal side of the blood capillaries, with labeling present in both plasma membranes and perinuclear cytoplasm (Figure 7F).

We then used double immunostaining to test whether Panx1 aggregates in lens plasma membrane were colocalized with connexin gap junction plaques. Multiplex confocal imaging did not reveal any significant colocalization between labeling specific to Cx50 and Panx1 in mouse lens slices (Figure 9). Sections made in the lens equatorial plane, which expose fibers sectioned across the length, revealed that punctate Panx1 labeling was evenly distributed along plasma membranes (Figure 9B,C). This is in contrast to the characteristic distribution of Cx50-labeled gap junctions, which form large plaques localized primarily to the broad sides of the hexagonal-shaped lens fibers. While connexins-specific labeling extended into the deep cortex, Panx1 staining decreased significantly in MF that had already completed elongation.

Discussion

In this work, we characterized the gene expression, localization and biochemical properties of the Panx1 and Panx2 gap junction proteins in the murine lens. Immunohistochemistry data on Panx1 was also obtained from blood capillaries attached to neonatal lenses. Our data showed the following: (1) expression of both pannexin genes is robust in the lens, with peak expression detected in the equatorial epithelium and apparent decline in elongated fibers; (2) Panx1 and Panx2 gene expression levels and protein localization patterns differed from that of Cx50; (3) Panx1 form small aggregates in the plasma membrane that do not colocalize with connexin gap junctions; (4) Panx1 are likely associated with the lipid raft sub-domains of plasma membranes.

Our RT-PCR and in situ hybridization data indicated that expression of both proteins in the lens is likely regulated developmentally. The in situ data showed that the peak expression of both genes was localized to the equatorial epithelium, just prior to differentiation into fibers. This was also confirmed by the immunohistochemistry in fixed lens slices. Due to lower sensitivity of the in situ technique Panx1 expression in elongated fibers is barely detectable. The sensitive technique, quantitative real time RT-PCR showed that the concentration of Panx1 and Panx2 transcripts declined in excess of 10 fold as lens epithelial cells resumed elongation and continued a decrease as they matured further. The pace of the pannexin mRNA decline was higher compared to Cx50, a major lens gap junction protein. The data on differentiation-induced down regulation of Panx1 transcript are in good agreement with the results of recent studies in mouse brain [9,14]. The intensity of the Panx1-specific immunostaining also peaked at the lens periphery and declined toward the cortex and nucleus. It is fair to note, however, that diminished immunolabeling in the center may also reflect insufficient antibody penetration into dense lens nucleus.

Our western blot experiments showed that lens cells contained three Panx1 isoforms, 43 kDa, 58 kDa, and 62 kDa, and the oligomeric 120 kDa one. All isoforms, except the 43 kDa one, showed association with cell membranes but their ratio in the membrane-enriched fraction varied significantly. As fiber differentiation progressed, plasma membranes accumulated gradually more of the 62 kDa isoform and the 120 kDa oligomer, while the quantity of the ubiquitous 58 kDa isoform decreased. The 43 kDa isoform, was previously reported to be a monomeric form of Panx1 [9,23] since it is close to the predicted size of Panx1. The ubiquitously expressed 58 kDa membrane bound isoform is 11 kDa larger then the predicted size. Original reports had not detected alternatively spliced variants of Panx1 mRNA, which may have served as an explanation for the striking difference in the molecular weight of the Panx1 isoforms [12]. Phosphorylation, which could significantly decrease protein mobility in gel, has been demonstrated for several connexins [18]. However, phosphorylation is unlikely to cause such a significant increase in the molecular weight of Panx1 despite the presence of putative phosphorylation sites reported earlier [23,30]. Glycosylation is another type of modification reported for transmembrane proteins [19]. Amino acid sequence analysis data predicted one N-glycosylation site in amino acid position 337, suggesting a plausible explanation for the Panx1 molecular weight increase. N-glycosylation has been previously demonstrated for the cytosolic c-terminal portion of the thrombopoeitin molecule [31]. A full complement of the three isoforms observed in the lens was not detected in muscle, blood endothelial and erythrocyte ghosts, but was present in the retina, and reported in the brain in the prior observations [23]. Taken together with the robust expression of Panx2 in the lens, brain and retina, these may indicate functional parallels for the role of pannexins in these ocular tissues.

As evidenced by the Western blot and differential fractionation data, the 58 kDa isoform and the oligomeric 120 kDa isoform of Panx1 are associated with the plasma membrane, while the 62 kDa protein is likely to be associated with Golgi apparatus and ER. In support, localization to the ER and function in Ca²⁺ homeostasis has been reported for Panx1 recently [13]. First, the two larger isoforms were quantitatively separated from the 62 kDa isoform in crude cell lysates by low speed centrifugation, which normally pellets cell debris enriched in plasma membranes but not sediment Golgi microsomes. Second, the western blot data for erythrocyte ghosts preparation that is devoid of cytoplasmic organelles also contained a single 58 kDa band and lacked the 62 kDa isoform. Finally, detergent extraction experiments showing resistance to Triton X-100 and susceptibility to MCD/Triton X100 treatments, indicated that Panx1 complexes are likely associated with cholesterol-rich membrane microdomains termed lipid rafts [27,29,32,33]. As shown previously for Cx43 and Cx50, the gap junction plaque-associated connexins, but not extra-junctional ones, were resistant to Triton X-100 extraction [3,34]. Such resistance is explained by association with enriched lipid rafts enriched with cholesterol and can be disrupted by MCD treatment rendering connexins susceptible to Triton X-100 extraction, similar to what was observed with Panx1 [29]. Immunostaining with anti-Panx1 antibodies showed a plaque-like punctate labeling that decorated the plasma membranes of elongating lens fibers and the blood capillaries of tunica vasculosa lentis. In contrast to the ordered distribution of connexin gap junction plaques localizing primarily to the broad sides of hexagonal-shaped lens fibers, small Panx1 aggregates were scattered randomly along the fiber cell membranes (Figure 9).

Taken together, our data indicate that the membrane-bound isoforms of Panx1 participate in the formation of the detergent-resistant membrane complexes and form visible aggregates. No colocalization between Panx1 and Cx50 has been detected, however, despite the similarity in biochemical properties of the two proteins. These suggest that pannexins and connexins associate with spatially different lipid raft microdomains in the plasma membrane. Functional mosaicism of lipid raft microdomains arguably represent a mechanism of spatial control of signaling through raft membranes [35]. Many connexins were shown to associate with lipid rafts enriched with caveolin-1 [33]; however, other types of lipid rafts that differ in lipid and protein content were also characterized [35,36]. Further experiments are required to test which subtype of lipid rafts will colocalize with Panx1.

Given that Panx1 is the major channel-forming protein in the pannexin family [9], we assumed that immunostaining with anti-Panx1 antibodies adequately reflected the distribution of pannexin channels in the lens. The pattern of Panx1 labeling changed significantly after newly differentiated fibers resumed elongation. Punctate membrane labeling replaced the localization to peri-nuclear region in the undifferentiated epithelium. It has been shown for Cx43 (and probably other connexins, too [37]) that the newly synthesized molecules are first assembled into hemichannels in the Golgi [33]; the hemichannels are then transported to the plasma membrane to form functional cell-to-cell channels. By analogy, these data and HeLa cells immunostaining data suggested that, early in differentiation, Panx1 undergoes processing and maturation in the ER and Golgi, and translocates into the membrane later.

Recent experiments have suggested that recombinant Panx1 can form hemichannels as well as gap junctions connecting closely opposed cells when expressed in oocytes and human cell lines [13,16,19]. Alternatively, the pannexin channels might have a function in organelle membranes, as suggested by recent findings showing that Panx1 is implicated in Ca²⁺ homeostasis in the ER [13]. Prior experiments showing a complete electrical uncoupling of lens fibers in Cx46/Cx50 double knockout mice [5,20], lends weight to the argument against a redundant junctional function for pannexins. This point of view has gained more support recently from studies in blood erythrocytes possessing pannexin hemichannels [38]. Although our results do not allow us to distinguish between these three potential functions of the pannexins in the lens, we argue that our data on tight association with plasma membrane suggest that at least a portion of Panx1 form hemichannels in membranes of fiber cells and adjacent blood capillaries.

Pannexin channels possess properties distinct from connexin-mediated hemichannels in the oocyte system [7,16,17,19], and might not, therefore, be redundant in maintaining lens metabolic homeostasis. The results of physiological experiments have demonstrated that Panx-1 hemichannels support passage of electrical currents and are most likely implicated in Ca²⁺ and ATP transport through the intracellular and plasma membranes [13,16,19,38]. This has led to a suggestion that Panx1 may represent an ATP pore, which is capable of passing ATP through the membrane in a gap junction-independent mechanism of Ca²⁺ wave propagation [16,17,19]. Although Ca²⁺ wave propagation has not been demonstrated for the lens cells, pannexin channels may represent a conduit for metabolic passage of Ca²⁺ and ATP in the lens. Significantly, our immunohistochemistry data revealed similar distinct Panx1-specific labeling in blood endothelial cells of the tunica vasculosa lentis and in the lens fibers. Such labeling was evident on the luminal side of the blood capillaries and, together with the data on Panx1 expression in erythrocyte membranes, corroborates well with the recently suggested role for Panx1 hemichannels in the control of nitric oxide-mediated vasodilation [19,38]. Pannexin hemichannels have been suggested to play a variety of functions such as control of vascular tone in response to ischemia and mechanical (shear) stress in erythrocyte and endothelial cell membranes [19,30,38], fiber cell volume regulation [7], in ischemic death of CNS neurons [39] and control of Ca²⁺ homeostasis in the reticulum [13]. In the avascular, oxygen-deprived lens tissue, ischemia-activated pannexin hemichannels may play a role in the metabolic and ionic exchange between fibers and media. The discovery of pannexins in the lens adds to the list of communication pathways supporting ionic and metabolic homeostasis of this avascular tissue, and allows us to suggest a distinct, nonredundant to connexin function in the membrane.

Acknowledgements

We thank Dr. S. Lukyanov for the kind gift of pEGFP-Panx1 plasmid, Dr. V. Slepak for expert advice in protein purification and critical reading of the manuscript, Dr. R.J. Cenedella for consultation on the lens protein fractionation, and Drs. G. Dahl, A. LaGier and V. Narayanan for the critical reading of the manuscript. This study was supported by NIH grants R01-EY14232 (V.S.), a Research to Prevent Blindness (RPB) Career Development Award (V.S.), an unrestricted grant to the Department of Ophthalmology from RPB, and an unrestricted grant P30-EY014801 to the Bascom Palmer Eye Institute.

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