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(Investigative Ophthalmology and Visual Science. 2005;46:3247-3255.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1504
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Regulation of Lens Cell-to-Cell Communication by Activation of PKC{gamma} and Disassembly of Cx50 Channels

Guido A. Zampighi,1 Ana M. Planells,2 Dingbo Lin,3 and Dolores Takemoto3

1From the Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, Los Angeles, California; the 2Department of Orthodontics and Pediatric Dentistry, UCLA School of Dentistry, Los Angeles, California; and the 3Department of Biochemistry, Kansas State University, Manhattan, Kansas.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 (Cx46) and connexin50 (Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase C{gamma} (PKC{gamma}) affects the number of channels composed of Cx46, Cx50, or both connexins.

METHODS. Whole rat lenses were treated with phorbol-12-myristate-13-acetate (TPA) to activate PKC{gamma} or the inactive analogue 4{alpha}-phorbol,12,13-didecaneote (PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze-fracture immunolabeling (FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKC{gamma} activity, connexin phosphorylation and coimmunoprecipitation.

RESULTS. Treatment with TPA activated PKC{gamma} and uncoupled the lens cortex by ~60%. PDD had no effect. Activation of PKC{gamma} decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKC{gamma} was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin (Cav)-1. In the membrane environment, PKC{gamma} phosphorylated Cx50 at serines and threonines and Cx46 only at threonines.

CONCLUSIONS. The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.

The lens is an avascular tissue that can maintain homeostasis even with its mass of highly elongated fiber cells, which are located far away from their blood supply. A model has been proposed to understand this singularity, in which circulating fluxes link the transport of ions and water to the movement of nutrients and waste products within the lens.1 The circulating fluxes are mediated by the interplay of two pathways: one created by the narrow extracellular space between cells (the paracellular pathway) and the other formed by specialized cell-to-cell channels that directly couple the cytoplasm of neighboring cells. These channels are composed of proteins of the connexin family that are situated transversely between the plasma membranes of opposing cells and are themselves assembled in two-dimensional plaques, called gap junctions.2 3 Although the establishment of the gap junction pathways is crucial for both the formation of a large spherical shape and the overall resultant transparency, they convey the implicit danger that localized damage of a single fiber could quickly propagate throughout the entire lens. Therefore, a longstanding challenge has been to unravel the mechanisms that regulate gap junction coupling in the superficial cortical and deeper nuclear fibers.

Numerous studies have demonstrated that gap junction coupling is altered by a myriad of cell signals including the intracellular concentration of Ca2+ and protons,4 5 6 phosphorylation,7 8 environmental factors,9 channel blockers,10 anesthetics,11 12 and growth factors.13 14 These signals regulate coupling by two fundamental mechanisms: they change either the properties of the single channel or the number of channels assembled in the gap junction plaques. Although channel opening and closing involves conformational changes at the connexin protein level, the number of channels can be altered by various other factors, including the synthesis of connexins, the trafficking of hemichannels to the plasma membrane, and the assembly of the latter into gap junction channels. To distinguish between these coupling regulatory mechanisms, one needs information about the functional state of the channels themselves as well as accurate estimates of the number of connexin channels and hemichannels in the plasma membrane. This latter information is difficult to obtain, because the principal difference between them is the state of assembly (dodecamers versus hexamers) of the connexin protein in the plasma membrane.15

Lens fiber cells express approximately equal molar amounts of Cx46 and Cx50.16 17 Yet, the distribution of the coupling is regional: Cx46 couples both the cortical and the nuclear fibers, whereas Cx50 couples only the cortical fibers.18 We took advantage of this regional disparity to estimate the number of Cx50 channels and hemichannels in coupled and uncoupled conditions. Functional uncoupling was induced by activating a classic PKC isoform, PKC{gamma}, with the tumor-promoting phorbol-12-myristate-13-acetate (TPA), which is known to act like the natural PKC activator, diacylglycerol. In addition, we used another phorbol ester, 4{alpha}-phorbol 12,13-didecanoate (PDD) as a control, which is structurally similar to TPA but is nonfunctional.

We found that treatment with TPA partially uncoupled (~60%) cortical fibers to allow the passage of the fluorescent dye Lucifer yellow, whereas PDD had no effect. The treatment activated PKC{gamma}, translocated the enzyme to membrane fractions and phosphorylated Cx46 and Cx50. Quantitative freeze-fracture immunolabeling (FRIL) indicated that activating the PKC{gamma} signal transduction mechanism disassembled ~95% of the Cx50 channels into hemichannels and induced circular microdomains with an extremely low density of particles that appeared as voids within gap junctions. We concluded that the uncoupling mediated by the activation of PKC{gamma} involves the preferential, or perhaps exclusive, disassembly of Cx50 channels into hemichannels. In addition, our observations raise the possibility that channel disassembly occurs in morphologically distinct microdomains in the plasma membrane that may correspond to planar lipid rafts.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Materials
Monoclonal antibodies against PKC{gamma} and Cav-1 were obtained from BD Biosciences (Palo Alto, CA); monoclonal mouse anti-Cx50 (amino acids 290–440) from Zymed Laboratories (South San Francisco, CA); polyclonal rabbit anti-phosphothreonine (pT) and anti-phosphoserine (pS) from Chemicon (Temecula, CA); polyclonal rabbit anti-Cx46 from Alpha Diagnostic International., Inc. (San Antonio, TX); nonspecific rabbit IgG and protein-agarose beads (A/G PLUS) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (HRP), and nonradioactive PKC assay system (PepTag) from Promega (Madison, WI); Dulbecco’s modified Eagle’s medium (DMEM; low glucose), gentamicin, and penicillin-streptomycin from Invitrogen-Life Technologies (Carlsbad, CA); sodium fluoride (NaF) from Fisher Scientific (Pittsburgh, PA); phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail from Sigma-Aldrich (St. Louis, MO); optimal cutting temperature (OCT) compound from Sakura Finetec (Torrance, CA); Alexa Fluor 568 goat anti-rabbit IgG (H+L), Lucifer yellow, rhodamine dextran, and antifade medium (SlowFade) from Molecular Probes (Eugene, OR); and TPA and PDD from CalBiochem (San Diego, CA).

Animals
Rats were 100-g (~6-week old), male and female Sprague-Dawley rats from Charles River Laboratories (Wilmington, MA). All experiments conformed to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and were performed according to an institutionally approved animal protocol. In all experiments, lenses were incubated in 25 ng/mL TPA or PDD at 37°C, followed by processing for subsequent assays.

Gap Junction Activity Dye-Transfer Assay
Whole-lens gap junction activity was analyzed by dye-transfer assay, as described previously.19 Six-week-old Sprague-Dawley rats were euthanatized by CO2, and the lenses were removed immediately after the death of the animal and washed in PBS. Lenses were incubated with 25 ng/mL TPA or the inactive analogue PDD for 15 minutes. PDD has similar membrane permeability but is not an activator of PKCs.20 Lucifer yellow (2.5 mg/mL in PBS) was microinjected as described,19 and the lenses were subsequently incubated in serum-free DMEM at room temperature for 30 minutes, to allow dye transfer. Rhodamine dextran (1%) was injected with Lucifer yellow and used as a control for nonspecific leakage. A total 126 nL of Lucifer yellow and rhodamine dextran was injected into the superficial cortical fibers (around 20 µm in depth) per injection site, with a microinjection apparatus (Nanoliter 2000; World Precision Instruments, Inc., Sarasota, FL). After incubation, the lenses were fixed in 2% paraformaldehyde, dissected, and mounted in 3% agar. The extent of dye transfer (in millimeters; diffusion distance of rhodamine-dextran subtracted from Lucifer yellow diffusion distance) as a measure of gap junction permeability in the lens was determined by confocal microscopy. Each experimental group contained six lenses and the distance of dye transfer was determined in six areas of the bow region of each lens in coded samples. Results are expressed as mean ± SEM with P ≤ 0.05.

PKC{gamma} Activity Assay
PKC{gamma} activity was analyzed using a kit (PepTag Assay; Promega).21 22 Briefly, equal protein amounts of whole-cell extracts from whole lens were immunoprecipitated with PKC{gamma} antisera at 4°C for 4 hours, as previously described.22 Immunoprecipitated PKC{gamma}/agarose bead complexes were incubated with a PKC reaction mixture according to the manufacturer’s instruction. Boiling the samples at 95°C for 5 minutes stopped the reactions, and the PKC{gamma} reaction products (fluorescent PepTag peptides) were resolved by agarose gel electrophoresis and visualized under UV light. The phosphorylated peptide bands were excised, and their fluorescence intensities were quantified by spectrophotometry, according to the manufacturer’s instructions. Results are expressed as the percentage of nontreated specific PKC{gamma} activity.

Sucrose Gradient Centrifugation
Whole lenses were extracted with cell-lysis buffer containing 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.1% protease inhibitor cocktail, 5 mM NaF, and 2 mM PMSF, and 1% Triton X-100 and incubated on ice for 30 minutes. Whole-cell lysates were mixed with an equal volume of 80% sucrose in Mes-NaCl buffer containing 25 mM Mes (pH 6.5), and 150 mM NaCl, 0.1% protease inhibitor cocktail, 5 mM NaF, and 2 mM PMSF and laid at the bottoms of 12-mL ultracentrifuge tubes. The samples were overlaid with 8 mL of a 5% and 35% continuous sucrose gradient in Mes-NaCl buffer and centrifuged at 245,000g for 22 hours at 4°C in a swinging bucket rotor (SW41 Ti; Beckman, Fullerton, CA). Fractions (1 mL each, 12 fractions total) were collected from the top of each gradient. Protein samples were precipitated with 10% trichloroacetic acid (TCA), separated in 10% SDS-PAGE and immunovisualized by Western blot analysis. Fractions 3 to 6 were pooled and used for the experiments. For the coimmunoprecipitation and phosphorylation assays, the fractions were sonicated in 0.1% SDS.

Immunoprecipitation Analysis
Fractions 3 to 6 from the sucrose density were immunoprecipitated with anti-Cx46, -Cx50, or -Cav-1 at 4°C for 4 hours, as described.13 The immunoprecipitate–agarose bead complexes were resolved by SDS-PAGE and visualized by Western blot with antisera to Cx46, Cx50, Cav-1, or PKC{gamma}.

Phosphorylation of Cx46 and Cx50 on Ser and Thr
Fractions 3 to 6 from the gradient were immunoprecipitated with anti-Cx46 or Cx50 at 4°C for 4 hours as described.22 The immunoprecipitate–agarose bead complexes were resolved by SDS-PAGE and visualized by Western blot analysis with antisera to pS, pT, Cx46, or Cx50.

Quantitative FRIL
We used equatorial fibers from the lenses of five rats (~100 g body weight). One lens was the control and the other was immersed in a solution containing 25 ng/mL TPA in mammalian Ringer’s solution for 15 minutes at 37°C. FRIL was performed as described previously.23 24 25 Briefly, bundles of fibers (5–7 fibers) were rapidly frozen (without chemical fixatives) and fractured in a freeze-fracture apparatus (JEOL, Peabody, MA). The cleaved surfaces were shadowed with platinum-carbon at 70° and carbon at 90°. The tissue underneath the replica was removed with SDS buffer. The SDS-treated replica was labeled with the anti-Cx50 monoclonal antibody (1:50 dilution in PBS with 0.1% BSA) and rabbit anti-mouse antisera that were visualized with 10 nm protein A gold particles (the "sandwich" method). The replicas were studied by electron microscope (model CM10 [Carl Zeiss Meditec, Inc., Thornwood, NY] and Tecnai 12 [Philips, Eindhoven, The Netherlands], equipped with a 2 k x 2 k charge-coupled device [CCD] camera; Gatan, Pleasanton, CA).

Quantification
We quantified >100 µm2 of fiber plasma membranes (protoplasmic [P] and external [E] faces) at a 25,000x magnification. The negatives were digitized with a scanner (ArtiScan 2500f; MicroTek, Redondo Beach, CA) or collected in digitized format with a digital 2 k x 2 k CCD camera (Gatan). The ImageJ software package (NIH; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-imageJ; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) was used to estimate (1) the density of intramembrane particles in the gap junction plaques, (2) the density of intra-membrane particles in the P face of the plasma membrane, (3) the density of anti-Cx50 gold complexes labeling the gap junction plaques, (4) the density of anti-Cx50 gold complexes labeling the P face of the plasma membrane, and (5) the density of anti-Cx50 gold complexes labeling the E face of the plasma membrane (nonspecific labeling24 25 ). The measurements are expressed as the mean ± SD. The labeling ratio (LR) was the ratio of the density of intramembrane particles and the density of anti-Cx50 gold complexes in the same region.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Free-Fracture Immunolabeling
A critical advantage of using FRIL is the ability to quantify independently the density of Cx50 channels (dodecamers) in gap junction plaques and Cx50 hemichannels (hexamers) in the plasma membrane of fibers posed in different coupling conditions. This ability arises from the fact that membranes fracture down the middle of the phospholipid bilayer into complementary P and E faces (Figs. 1 2) . Proteins that traverse the phospholipid bilayer appear in these fracture faces as distinct intramembrane particles, and those composed of Cx5024 25 26 27 are labeled with antibody gold complexes. There is, however, a significant distinction between the labeling of Cx50 assembled in gap junction channels and that in the hemichannels of the plasma membrane. Because the SDS treatment does not remove the unfractured membrane of the opposing fiber, both the P and E fracture faces of the gap junction plaque are labeled with gold complexes.27 Consequently, the density of gold complexes on the P and E faces of the gap junction determines the number of Cx50 channels (Figs. 1 2) . In contrast, when a gap junction plaque is not present, the SDS treatment removes the plasma membrane of the opposing fiber in the non–gap-junction plaques regions. Therefore, only the density of gold complexes labeling the P face of the plasma membrane determines the number of Cx50 hemichannels (Fig. 3) . Because the complementary E face of the plasma membrane contains the impression of the hemichannel in the ice (the E face "pit"), the density of gold complexes in this face determines the nonspecific binding.24 25



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  		FIGURE 1. Labeling of a gap junction plaque with anti-Cx50 gold complexes. The fracture plane revealed the E face of one fiber and the P face of the opposing fiber. The gap junction appeared as a plaque of particles in the P face and complementary pits in the E face. The height of the step separating the faces (the E-to-P transition) corresponds to the width of the extracellular space. In the gap junction, the E-to-P transition exhibited little height (arrowheads) indicating a narrow extracellular gap separating the membranes. Outside the gap junction plaque, the E-to-P transition contained a higher fracture step, indicating a wider extracellular space (arrows). Because the SDS treatment does not remove the opposing junctional membrane, the anti-Cx50 gold complexes (~10 nm diameter dark discs) labeled both faces.

 


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  		FIGURE 2. Distribution of anti-Cx50 gold complexes in the plasma membrane of untreated fibers. In this low-magnification view (~50 µm2), the fracture plane exposed extensive areas of P and E faces of cortical fibers to antibody labeling. The area contains >80 gap junction plaques that were labeled with anti-Cx50 gold complexes at high density. In an effort to simplify their visualization, the gap junction plaques were colored reddish.

 


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  		FIGURE 3. Distribution of anti-Cx50 gold complexes in the nonjunctional plasma membrane of cortical fibers. The view shows regions of both P and E faces. The P face contained gap junction plaques, labeled at high density (lower border), and nonjunctional plasma membranes, labeled at lower density. Note that the complexes labeling the P face of the nonjunctional plasma membrane appear as loose clusters, often located at the boundaries of microdomains (inset). The E face was labeled with an even lower density of complexes.

 
Cx50 Channels and Hemichannels in Untreated Fibers
Before TPA treatment, the gap junction plaques had 8200 ± 1200 particles/µm2, a density corresponding to the sum of the Cx50 and the Cx46 channels. The P and E faces of the plaques were labeled with 578 ± 122 anti-Cx50 gold complexes/µm2, a density ~30 times higher than the nonspecific labeling estimated from the labeling of the E fracture faces (~18/µm2; Table 1 ). The ratio of unlabeled particles to gold complexes (the "labeling ratio") indicated that there was 1 anti-Cx50 gold complex for every ~15 particles in the plaques. Because fiber gap junctions contain channels composed of Cx46 and Cx50,16 17 the labeling ratio for Cx50 should be significantly higher than the one estimated from the total density of particles assembled in gap-junction plaques.


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  		TABLE 1. Labeling Densities

 
The P face of the fiber plasma membrane, in contrast, had 2087 ± 194 particles/µm2, which represent the normal complement of integral proteins spanning the plasma membrane (Figs. 1 2 3 ; Table 1 ). This P face was labeled with 33 ± 10 anti-Cx50 gold complexes/µm2, which is ~17 times lower than the density of labeling in gap junctions and ~1.8 times higher than that of the nonspecific labeling estimated from the E faces (Table 1) . The ratio of these densities indicated that there was 1 anti-Cx50 gold complex for every ~63 intramembrane particles in the fiber plasma membrane. Unexpectedly, we observed that often these anti-Cx50 gold complexes appeared as clusters (Fig. 3) at the boundaries of circular microdomains with a very low density of intramembrane particles (Fig. 3 , inset).

Normalizing the densities of anti-Cx50 complexes labeling the surface of an average fiber of 2000/µm2 of surface area, 5% of which is occupied with gap junctions24 25 gave rise to the prediction that the number of Cx50 channels in the gap junctions was approximately equal to the number of Cx50 hemichannels in the plasma membrane.

Cx50 Channels and Hemichannels in Treated Fibers
Treatment with TPA altered the overall morphology of gap junctions, decreasing the density of intramembrane particles as well as the density of the anti-Cx50 gold complexes labeling them (Fig. 4 ; Table 1 ). The density of particles assembled in plaques decreased by a factor of ~3 (from 8500 ± 1500/µm2 in control to 2982 ± 1033/µm2 after treatment) and the density of anti-Cx50 gold complexes by a factor of ~9 (from 578 ± 122/µm2 to 64 ± 15/µm2). Therefore, the labeling ratio decreased from 1:13 to 16 to ~1:50 after treatment with TPA. The simultaneous decrease in the density of particles in plaques and the anti-Cx50 gold complexes labeling them indicated that the number of Cx50 channels decreased on TPA treatment.



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  		FIGURE 4. Distribution of anti-Cx50 gold complexes in TPA-treated fibers. Treatment with TPA altered the shape of gap junctions and decreased the density of gold complex labeling. The gap junctions appeared either as irregular plaques with circular areas exhibiting an extremely low density of particles (left side), or as strings of particles shaped as stars (top right quadrant). The decrease in the density of gold complexes was determined by quantification (Table 1) . Note that the location of the gold complexes shifted toward the plaque’s boundaries. Black disks were superimposed on the gold complexes to help visualize this shift toward the nonjunctional plasma membrane.

 
A noteworthy feature of the fibers treated with TPA was the appearance of circular microdomains within the gap junction plaques that appeared as "voids" and exhibiting an extremely low density of intramembrane particles (Figs. 4 5B 5C) . These voids were curved toward the cytoplasm ("negative" curvature) and exhibited diameters ranging from 22 to >300 nm. The largest voids were encircled by strings of intramembrane particles that were either poorly labeled or not labeled at all by the anti-Cx50 gold complexes (Figs. 4 5D) . Also, the density of particles and anti-Cx50 gold complexes increased, albeit slightly, from 33 ± 10 to 58 ± 8/µm2 in the plasma membrane (Table 1) .



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  		FIGURE 5. Comparison of untreated and TPA-treated gap junctions. (A) A gap junction from an untreated fiber composed of intramembrane particles was arranged in a dense plaque, labeled at high density (black disks). (B) Some plaques exhibited small circular microdomains, labeled at extremely low density (red regions). (C) Treatment with TPA increased the diameter of the microdomains and decreased the density of the anti-Cx50 gold complexes (Table 1) . (D) In some regions of the plasma membrane, the morphology of the gap junction changed from a dense plaque (A, B) to star-shapes composed of strings containing double or single intramembrane particles. These highly modified plaques were labeled with the lowest density of anti-Cx50 gold complexes.

 
Normalizing the densities of antibody gold complexes to an average fiber of 2000/µm2 surface area suggested that the distribution of anti-Cx50 complexes changed from being approximately equal between the gap junction plaques and the plasma membrane, to being overwhelmingly (~95%) in the fiber plasma membrane.

The changes induced by TPA in gap junction morphology and labeling densities were not observed in fibers located deeper within the lens, because exposure to TPA lasted only10 to 15 minutes, which is not long enough for the penetration of TPA in these fibers. The lack of change in gap junction labeling densities in these deeper regions thus represents an additional control and insures that the changes ascribed to TPA treatment are attributed correctly (Fig. 5) .

It thus seems clear that treatment with TPA disassembled the Cx50 channels into hemichannels, leaving the unlabeled channels (probably composed of Cx46) grouped in irregularly shaped gap junction plaques containing lipid microdomains.

Fiber Coupling
To determine whether the decrease in the density of anti-Cx50 gold complexes in plaques translated into fiber uncoupling, we measured diffusion of the dye Lucifer yellow in untreated lens and after treating them with TPA and PDD for 30 minutes (Fig. 6) . In untreated lenses, the dye moved ~140 µm into the lens, a distance corresponding to ~28 layers of fibers. After TPA treatment, the dye diffused only ~40 µm into the cortex, corresponding to ~11 layers (Fig. 6 ; TPA). Because the diffusion of Lucifer yellow into the cortex remained unaltered by treating lenses with the permeable but nonfunctional phorbol ester PDD,20 we concluded that the changes induced by TPA do not include changes in the permeability of the plasma membrane.



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  		FIGURE 6. Dye coupling decreased with TPA treatment. (A) Confocal microscopy was used to measure the depth of Lucifer yellow dye transfer (in micrometers) from the point of injection in the equatorial region of lenses. The movement of the dye through the extracellular spaces was accounted for by subtracting the rhodamine-dextran fluorescence. Each experimental group contained six lenses, and the results are expressed as mean ± SEM. *Significant difference at P ≤ 0.05. (B) Images of an untreated (Cont) lens and PDD- and TPA-treated lenses are shown, to demonstrate the diffusion of the dyes within the lens. Green: Lucifer yellow; yellow: represents the merged fluorescence of the rhodamine dextran and Lucifer yellow. Red arrows: the dye microinjection site and the direction of dye transfer; red bars: distance of dye transfer into the lenses.

 
Activation of PKC{gamma} and Phosphorylation of Cx46 and Cx50
Enzyme activity assays showed that the activity of PKC{gamma} increased in lenses treated with TPA, but not in lenses treated with PDD (Fig. 7) . To determine whether PKC{gamma} activation phosphorylates the connexins, we performed a series of immunoprecipitation experiments (Fig. 8) . First, immunoprecipitation with Cav-1 indicated that significant amounts of PKC{gamma} became associated with membrane fractions in lenses treated with TPA, though not with those treated with PDD (Fig. 8A ; lane 1). The distribution of Cx46 and Cx50 in these same fractions remained unaltered by the TPA treatment (Fig. 8A) . We then tested for connexin phosphorylation by immunoprecipitating membrane fractions with antibodies against Cx46 and Cx50 and probing them with antibodies against phospho-serines and phospho-threonines (Fig. 8B) . We observed that both connexins were phosphorylated, though with one significant difference: Cx50 was phosphorylated at serine and threonine residues, whereas Cx46 was phosphorylated only at threonines (Fig. 8B) . The serines in Cx46 were maximally phosphorylated in untreated and control lenses treated with PDD.



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  		FIGURE 7. PKC{gamma} activity increased with TPA treatment. Membrane fractions isolated from untreated and TPA- or PDD-treated lenses were immunoprecipitated with anti-PKC{gamma}. The enzymatic activity was measured with the PKC peptide substrate, C1 (PepTag; Pomega, Madison, WI). The data were normalized by calibrating the relative level of phosphorylated C1 substrate to the relative amount of PKC{gamma} in the immunoprecipitate, as determined by Western blot analysis. Enzyme activity was expressed as a percentage and the activity measured in untreated fractions was made equal to 100%. The experiments were performed in triplicate. Data are the mean ± SEM *Significant increase at P ≤ 0.05.

 


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  		FIGURE 8. (A) Coimmunoprecipitation of PKC{gamma} with Cx46, Cx 50, or Cav-1. Activated PKC{gamma} was translocated to fractions containing Cx46 and Cx50. Lens plasma membrane fractions were immunoprecipitated with anti-Cav-1 and the complexes analyzed by Western blot with anti-Cx46 and -Cx50 or -PKC{gamma}. Note that detectable levels of PKC{gamma} translocation into membrane fractions occurred only in membranes from TPA-treated lenses. In contrast, treatment with TPA did not alter the distribution of Cx46 or Cx50 in the fractions. (B) Phosphorylation of Cx50 and Cx46 by TPA. Activated PKC{gamma} phosphorylates Cx46 and Cx50. To determine connexin phosphorylation, membrane fractions of untreated and treated lenses were precipitated with anti-Cx50 and -Cx46 and then probed with antibodies against phosphoserines and phosphothreonines. Treatment with TPA induced phosphorylation of Cx50 at both serines and threonines but induced phosphorylation of Cx46 only at threonines. Treatment with the nonfunctional phorbol ester PDD had no effect. The experiments were performed in triplicate and nonspecific rabbit IgG was used as a negative control (NS). Blots loaded with antibodies against Cx46 or Cx50 served as the loading control.

 
We concluded, therefore, that treatment with TPA triggers a cascade of events in cortical fibers involving PKC{gamma} activation, translocation to the plasma membrane, connexin phosphorylation, Cx50 channel disassembly, and partial fiber uncoupling.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The principal conclusion of our studies is that the activation of PKC{gamma} in cortical fibers regulates the number of Cx50 and Cx46 channels independently. Experimental support for this conclusion includes: (1) the partial (~60%) uncoupling of fibers as determined from the decrease in the transfer of Lucifer yellow (Fig. 6) ; (2) the decrease in the density of intramembrane particles in gap junction plaques and of the anti-Cx50 gold complexes labeling them (Table 1) ; (3) the increase in the density of anti-Cx50 gold complexes labeling the fiber plasma membrane (Table 1) ; (4) the appearance of distinct circular voids within gap junction plaques (Figs. 4 5C 5D) ; and (5) the translocation of PKC{gamma} to the plasma membrane and subsequent phosphorylation of Cx46 and Cx50 (Fig. 8) . Taken together, these experimental observations indicate that the activation of PKC{gamma} disassembles the Cx50 channels into hemichannels.

What is the functional significance of the independent regulation of the number of Cx50 and Cx46 channels? Because fibers express Cx46 and Cx50 in approximately equal molar amounts,17 it has been assumed that each connexin isoform should account for one half of the coupling between fibers. Yet, genetic ablation has demonstrated that Cx46 is responsible for 100% of the coupling between nuclear fibers and a predominant amount of the coupling between cortical fibers.18 Currently, Cx50 is thought to play a role in lens development, since its ablation induces microphthalmia.28 29 Yet, the observation that activation of PKC{gamma} causes the preferential disassembly of the Cx50 channels supports the conclusion that Cx50 plays a role in maintaining the homeostasis of the mature lens as well. Our working hypothesis is that in the lens cortex—the region where Cx50 is functionally relevant—localized uncoupling can circumvent damaged fibers without impairing the patency of the pathway between the metabolically active fibers in the lens surface and the interior.

Given the disassembly of Cx50 channels, the question arises of whether the resulting hemichannels remain in the plasma membrane or are instead retrieved into the cytoplasm for recycling or degradation. One argument that the Cx50 hemichannels remain in the plasma membrane was derived from calculations of the total number of labeled particles after normalizing to a 2000/µm2 area. These calculations indicate that before and after TPA treatment, the total number of labeled particles remained constant (~121,000 in controls and ~117,000 after treatment), despite a 90% decrease in the labeled particles assembled in plaques. The most likely interpretation is that after channel disassembly, the Cx50 hemichannels do not become internalized but instead remain within the plasma membrane.

Why is activation of the PKC{gamma} cascade less efficient in regulating the number of Cx46 channels? Previous studies in cultured lens anterior epithelial cells indicate that PKC phosphorylation of connexin-43 (Cx43) at S368 uncouples gap junctions.30 Cx50 differs from Cx43 in a single amino acid residue in the domain of SKAS430SRA for Cx50 and SRAS368SRA for Cx43, whereas the amino acid sequence of Cx46 in this region is completely different. Although these differences in sequence can explain why PKC{gamma} phosphorylates serines in Cx50 but not in Cx46 (Fig. 8B) , they do not explain why the serines in Cx46 are already phosphorylated31 in the lenses used as the control (Fig. 8B) . It seems likely that the phosphorylation of serines in Cx46 involves different kinases, a hypothesis supported by the presence of recognition sequences for PKA and MAPK in the Cx46 sequence. A question that remains unanswered, however, is why phosphorylation of serines in a particular domain of the Cx46 sequence (around S430) disassembles the Cx50 channels but phosphorylation of serines in other domains leaves these channels structurally and functionally intact. Access to the atomic structure might eventually shed light on this crucial property of these channel-forming proteins.

The relationship between connexins, caveolins, and lipid rafts has been studied using heterologous expression in human embryonic kidney 293T and Cos-7 cultured cells.32 A principal conclusion of these studies is that partition within rafts is connexin specific. In particular, Cx50 appeared to be excluded from the lipid rafts, since it was not immunoprecipitated with Cav-1.32 Our observation that, on activation of PKC{gamma}, the disassembly of Cx50 channels induced distinct microdomains within the remaining gap junction plaques supported this hypothesis (Figs. 4 5) . Yet, observations such as the presence of Cx50 hemichannels at the edges of circular microdomains in the fiber plasma membrane (Fig. 3) and the colocalization of Cav-1, Cx46, and Cx50 in the same membrane fractions contradicted this hypothesis (Fig. 8A) . Perhaps these discrepancies underline peculiar properties of lens fiber cells, such as their extremely high content of cholesterol and their small number of membrane invaginations containing caveolins (caveolae). Therefore, it appears that to determine the relationship between connexins and lipid rafts, one must also include measurements in tissues where these specific connexins are endogenously expressed.

In conclusion, combining structural, functional, and chemical information, we have determined that functional uncoupling mediated by the activation of PKC{gamma} involves the phosphorylation of both Cx46 and Cx50, but preferential, or perhaps exclusive, disassembly of Cx50 channels into hemichannels. In addition, our observations raise the possibility that channel disassembly occurs in morphologically distinct microdomains in the plasma membrane that might correspond to planar lipid rafts. Finally, our study provides experimental support for the hypothesis that gap junctions composed of mixtures of Cx46 and Cx50 channels provide malleable pathways that can circumvent damaged regions while maintaining the patency of the pathways between the lens nucleus and the metabolically active fibers near the surface.


   Acknowledgements
 
The authors thank Ernest Wright for many suggestions during the preparation of the manuscript.


   Footnotes
 
Supported by National Eye Institute Grants EY-040110 and EY-13421 (DT), the Foundation to Prevent Blindness (GAZ), and the National Organization for Rare Disorders (DL).

Submitted for publication December 20, 2004; revised April 1 and 25, 2005; accepted July 12, 2005.

Disclosure: G.A. Zampighi, None; A.M. Planells, None; D. Lin, None; D. Takemoto, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Guido A. Zampighi, Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, Los Angeles, CA 90095-1763; gzampighi{at}mednet.ucla.edu.


   References
Top
 Abstract
 Methods
 Results
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 References
 

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