Identification of a nonselective cation channel in isolated lens fiber cells that is activated by cell shrinkage

2012 ◽  
Vol 303 (12) ◽  
pp. C1252-C1259 ◽  
Author(s):  
S. J. Gunning ◽  
K. K. H. Chung ◽  
Paul J. Donaldson ◽  
K. F. Webb
Keyword(s):  
Short Fiber ◽  
Membrane Properties ◽  
Lens Fiber ◽  
Cell Shrinkage ◽  
Patch Clamp Technique ◽  
Hypertonic Stress ◽  
Fiber Cells ◽  
Cation Conductance ◽  
Normal Lens ◽  
Lens Physiology

The initiation of lens cataract has long been associated with the development of a membrane “leak” in lens fiber cells that depolarizes the lens intracellular potential and elevates intracellular Na+ and Ca2+ concentrations. It has been proposed that the leak observed in cataractous lenses is due to the activation of a nonselective cation (NSC) conductance in the normal electrically tight fiber cells. Studies of the membrane properties of isolated fiber cells using the patch-clamp technique have demonstrated a differentiation-dependent shift in membrane permeability from K+-dominated in epithelial and short fiber cells toward larger contributions from anion and NSC conductances as fiber cells elongate. In this study, the NSC conductances in elongating lens fiber cells are demonstrated to be due to at least two distinct classes: a Gd3+-sensitive, mechanosensitive channel whose blockade is essential for obtaining viable isolated fiber cells, and a second Gd3+-insensitive, La3+-sensitive conductance that appears to be activated by cell shrinkage. This second conductance was eliminated by the replacement of extracellular Na+ with the impermeant cation N-methyl- d-glucamine and was potentiated by both hypertonic stress and isosmotic cell shrinkage evoked by the replacement of extracellular Cl− with the impermeant anion gluconate. This additional cation conductance may play a role in normal lens physiology by mediating regulatory volume increase under osmotic or other physiological challenges. Since the inappropriate activation of NSC channels is implicated in the initiation of lens cataract, they represent potential targets for the development of novel anticataract therapies.

scholarly journals Cx46 hemichannels contribute to the sodium leak conductance in lens fiber cells

2014 ◽  
Vol 306 (5) ◽  
pp. C506-C513 ◽  
Author(s):  
Lisa Ebihara ◽  
Yegor Korzyukov ◽  
Sorabh Kothari ◽  
Jun-Jie Tong
Keyword(s):  
Open Channel ◽  
Basal Membrane ◽  
Resting Potential ◽  
Channel Noise ◽  
Lens Fiber ◽  
Patch Clamp Technique ◽  
Fiber Cells ◽  
Whole Cell Patch Clamp ◽  
Ionic Fluxes ◽  
Persistent Inward Current

The lens is proposed to have an internal microcirculation system consisting of continuously circulating ionic fluxes that play an essential role in maintaining lens transparency. One of the key components of this system is the sodium leak conductance. Here we investigate the contribution of Cx46 hemichannels to the basal membrane permeability of peripheral fiber cells isolated from transgenic mouse lenses lacking Cx50 or both Cx50 and Cx46 (dKO) using the whole cell patch-clamp technique. Our results show that Cx46 hemichannels were largely closed at a resting voltage of −60 mV in the presence of millimolar divalent cation concentrations. However, even though the vast majority of these channels were closed at −60 mV, a small, persistent, inward current could still be detected. This current could be mostly blocked by exposure to 1 mM La3+ and was not observed in fiber cells isolated from dKO mouse lenses suggesting that it was due to Cx46 hemichannels. In addition, Cx50−/− fiber cells showed increased open channel noise and a depolarized resting potential compared with dKO fiber cells. Exposure of Cx50−/− fiber cells to La3+ hyperpolarized the resting potential to −58 mV, which is similar to the value of resting potential measured in dKO fiber and significantly reduced the open channel noise. In conclusion, these results suggest that Cx46 hemichannels may contribute to the sodium leak conductance in lens fiber cells.

Calmodulin-mediated gating of lens gap junction channels in vesicles

1984 ◽  
Vol 42 ◽  
pp. 134-137
Author(s):  
Camillo Peracchia ◽  
Stephen J. Girsch
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Orthogonal Arrays ◽  
Eye Lens ◽  
Lens Fiber ◽  
Cell Coupling ◽  
Particle Spacing ◽  
Fiber Cells ◽  
Gap Junction Channels ◽  
Communicating Junctions

The fiber cells of eye lens communicate directly with each other by exchanging ions, dyes and metabolites. In most tissues this type of communication (cell coupling) is mediated by gap junctions. In the lens, the fiber cells are extensively interconnected by junctions. However, lens junctions, although morphologically similar to gap junctions, differ from them in a number of structural, biochemical and immunological features. Like gap junctions, lens junctions are regions of close cell-to-cell apposition. Unlike gap junctions, however, the extracellular gap is apparently absent in lens junctions, such that their thickness is approximately 2 nm smaller than that of typical gap junctions (Fig. 1,c). In freeze-fracture replicas, the particles of control lens junctions are more loosely packed than those of typical gap junctions (Fig. 1,a) and crystallize, when exposed to uncoupling agents such as Ca++, or H+, into pseudo-hexagonal, rhombic (Fig. 1,b) and orthogonal arrays with a particle-to-particle spacing of 6.5 nm. Because of these differences, questions have been raised about the interpretation of the lens junctions as communicating junctions, in spite of the fact that they are the only junctions interlinking lens fiber cells.

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens

Development ◽  
1992 ◽  
Vol 115 (3) ◽  
pp. 813-820
Author(s):  
L.L. Harris ◽  
J.C. Talian ◽  
P.S. Zelenka
Keyword(s):  
Cell Proliferation ◽  
Protein Product ◽  
Mrna Levels ◽  
Lens Fiber ◽  
Proliferating Cells ◽  
Fiber Cells ◽  
Embryonic Chicken ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.

Cloning and functional expression of an MIP (AQP0) homolog from killifish (Fundulus heteroclitus) lens

2001 ◽  
Vol 281 (6) ◽  
pp. R1994-R2003 ◽  
Author(s):  
Leila V. Virkki ◽  
Gordon J. Cooper ◽  
Walter F. Boron
Keyword(s):  
Water Permeability ◽  
Fundulus Heteroclitus ◽  
Water Channel ◽  
Fold Increase ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes ◽  
Lens Fiber ◽  
Fiber Cells ◽  
Aqp1 Expression ◽  
Very High

The major intrinsic protein (MIP) of lens fiber cells is a member of the aquaporin (AQP) water channel family. The protein is expressed at very high levels in lens fiber cells, but its physiological function is unclear. By homology to known AQPs, we have cloned a full-length cDNA encoding an MIP from the lens of killifish ( Fundulus heteroclitus). The predicted protein (263 amino acids; GenBank accession no. AF191906 ) shows 77% identity to amphibian MIPs, 70% identity to mammalian MIPs, and 46% identity to mammalian AQP1. Expression of MIPfun in Xenopus laevis oocytes causes an ∼40-fold increase in oocyte water permeability. This stimulation is comparable to that seen with AQP1 and substantially larger than that seen with other MIPs. The mercurials HgCl2 and p-chloromercuribenzenesulfonate inhibit the water permeability of MIPfun by ∼25%. MIPfun is not permeable to glycerol, urea, or formic acid but is weakly permeable to CO2.

scholarly journals The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.

1994 ◽  
Vol 14 (10) ◽  
pp. 6743-6754 ◽  
Author(s):  
L Fromm ◽  
W Shawlot ◽  
K Gunning ◽  
J S Butel ◽  
P A Overbeek
Keyword(s):  
Cell Cycle ◽  
Transgenic Mice ◽  
Retinoblastoma Protein ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
T Antigen ◽  
Lens Fiber ◽  
Large T Antigen ◽  
Fiber Cells

Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens

2008 ◽  
Vol 294 (5) ◽  
pp. C1133-C1145 ◽  
Author(s):  
Kevin F. Webb ◽  
Paul J. Donaldson
Keyword(s):  
Large Population ◽  
Reversal Potential ◽  
Membrane Resistance ◽  
Cellular Level ◽  
Membrane Properties ◽  
Nonselective Cation Channel ◽  
Impedance Measurements ◽  
Fiber Cells ◽  
Anion Conductance ◽  
Ion Substitution

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd3+. Isolated fiber cells were viable in physiological [Ca2+] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens ( 1 ). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 μm) contained variable combinations of Ba2+-and tetraethylammonium-sensitive K+ currents. Longer fiber cells (150–650 μm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na+ with N-methyl-d-glucamine, indicating that the lens contains both Gd3+-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K+ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate.

The distribution of the fiber cell intrinsic membrane proteins MP20 and connexin46 in the bovine lens

1992 ◽  
Vol 103 (1) ◽  
pp. 245-257 ◽  
Author(s):  
E. Tenbroek ◽  
M. Arneson ◽  
L. Jarvis ◽  
C. Louis
Keyword(s):  
Monoclonal Antibody ◽  
Fiber Cell ◽  
Immunogold Electron Microscopy ◽  
Lens Fiber ◽  
Lens Fiber Cell ◽  
Western Blots ◽  
Fiber Cells ◽  
Bovine Lens ◽  
Narrow Side ◽  
Lens Membranes

MP20 is an intrinsic membrane protein previously identified in mammalian lens fiber cells. To identify a possible role for this protein in the lens, the distribution of MP20 and connexin46 has now been examined. Western immunoblotting with an anti-peptide antibody generated to the C-terminal 8 amino acids of MP20 confirmed the presence of this protein in the lens of several different mammalian species. A monoclonal antibody 5H1 was prepared that, in Western blots of bovine lesn membranes, recognized the same component as an antibody to rat connexin46 (Cx46). The apparent molecular mass of this component decreased from 59 kDa to 55 kDa following treatment of lens membranes with alkaline phosphatase. A monoclonal antibody to connexin-related MP70 recognized a 70 kDa component in bovine lens membranes confirming the presence of these two different connexin proteins in bovine lens membranes. To localize MP20 and Cx46 in the bovine lens membrane, lens fiber cell bundles were immunofluorescently labeled with both the MP20 antibody, and the monoclonal antibody to Cx46. Cx46 was identified in large plaques on the broad faces of the lens fiber cells throughout the outer 1 mm of the lens cortex. MP20 colocalized with Cx46 only in a restricted area 0.5 mm to 1.0 mm into the lens. In other regions of the lens, MP20 appeared more diffusely distributed over the fiber cell surface, although apparently concentrated in the ball-and-socket regions at the corners of the narrow side of the inner cortical lens fiber cells. These inner cortical regions were devoid of Cx46. A difference in distribution of these two proteins was confirmed in studies of immunofluorescently labeled lens cryosections. Furthermore, immunogold electron microscopy of purified lens membranes identified MP20 in both junctional regions (with Cx46) and in single membranes. These results provide evidence for a role for MP20 in mammalian lens fiber cell junctional formation or organization.

Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells

1992 ◽  
Vol 103 (3) ◽  
pp. 709-718 ◽  
Author(s):  
M. Brunkener ◽  
S.D. Georgatos
Keyword(s):  
Binding Capacity ◽  
Lens Fiber ◽  
Alkaline Ph ◽  
Trypsin Treatment ◽  
Binding Properties ◽  
Fiber Cells ◽  
Lens Membranes ◽  
Acylated Proteins ◽  
Triton X

Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a ureaextractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least two-fold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2 × 10(−7) and 4 × 10(−7) M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology

2020 ◽  
Vol 29 (12) ◽  
pp. 2076-2097 ◽  
Author(s):  
Carrie E Barnum ◽  
Salma Al Saai ◽  
Shaili D Patel ◽  
Catherine Cheng ◽  
Deepti Anand ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Morphology ◽  
Fiber Cell ◽  
Maturation Stage ◽  
Lens Fiber ◽  
Protein Fluorescence ◽  
Pediatric Cataract ◽  
Fiber Cells ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Degradation

Abstract Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7−/− lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7–ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

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