33 results on '"White, Thomas A."'
Search Results
2. Voltage Gating Properties of Channels Formed by a Skate Retinal Connexin
- Author
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White, Thomas W., Ripps, Harris, Srinivas, Miduturu, and Bruzzone, Roberto
- Published
- 2000
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3. Targeted Ablation of Connexin50 in Mice Results in Microphthalmia and Zonular Pulverulent Cataracts
- Author
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White, Thomas W., Goodenough, Daniel A., and Paul, David L.
- Published
- 1998
4. Selective Interactions among the Multiple Connexin Proteins Expressed in the Vertebrate Lens: The Second Extracellular Domain Is a Determinant of Compatibility between Connexins
- Author
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White, Thomas W., Bruzzone, Roberto, Wolfram, Sigrid, Paul, David L., and Goodenough, Daniel A.
- Published
- 1994
5. Unique and Redundant Connexin Contributions to Lens Development
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White, Thomas W.
- Published
- 2002
6. Differential regulation of Connexin50 and Connexin46 by PI3K signaling.
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Martinez, Jennifer M., Wang, Hong-Zhan, Lin, Richard Z., Brink, Peter R., and White, Thomas W.
- Subjects
PHOSPHOINOSITIDES ,CONNEXINS ,CELLULAR signal transduction ,KINASES ,GENETIC regulation ,GAP junctions (Cell biology) - Abstract
Gap junction channels can modify their activity in response to cell signaling pathways. Here, we demonstrate that Connexin50 (Cx50) coupling, but not Connexin46 (Cx46), increased when co-expressed with a constitutively active p110α subunit of PI3K in Xenopus oocytes. In addition, inhibition of PI3K signaling by blocking p110α, or Akt, significantly decreased gap junctional conductance in Cx50 transfected HeLa cells, with no effect on Cx46. Alterations in coupling levels were not a result of reduced Cx50 unitary conductance, suggesting that changes in the number of active channels were responsible. These data indicate that Cx50 is specifically regulated by the PI3K signaling pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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7. The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity.
- Author
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Mhaske, Pallavi V., Levit, Noah A., Li, Leping, Hong-Zhan Wang, Lee, Jack R., Zunaira Shuja, Brink, Peter R., and White, Thomas W.
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GENETIC mutation ,CONNEXINS ,ION channels ,SKIN disease diagnosis ,KERATITIS ,ICHTHYOSIS ,GENETICS of deafness ,GENE expression ,GENETICS - Abstract
Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26- A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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8. Lens Gap Junctions in Growth, Differentiation, and Homeostasis.
- Author
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Mathias, Richard T., White, Thomas W., and Xiaohua Gong
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GAP junctions (Cell biology) , *PLANT cells & tissues , *CONNEXINS , *LENS (Plants) , *CELL communication , *PLANT growth - Abstract
The article presents a study on lens gap junctions. It states that a gap junction is made up of various types of intercellular channels and its formation depends on the compatibility of connexins with each other. It mentions that SBFI, a Na+ indicator dye, was injected to test the effect of knocking out (KO) GPX-1 on lens circulation. The results show that an increase on Na+ and Ca2+ concentrations were seen along with the reductions in coupling conductance.
- Published
- 2010
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9. Connexin26 deafness associated mutations show altered permeability to large cationic molecules.
- Author
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Meşe, Gühistan, Valiunas, Vírginijus, Brink, Peter R., and White, Thomas W.
- Subjects
HOMEOSTASIS ,DEAFNESS ,CONNEXINS ,MEMBRANE proteins ,PHYSIOLOGY ,PHYSIOLOGICAL control systems - Abstract
Intercellular communication is important for cochlear homeostasis because connexin26 (Cx26) mutations are the leading cause of hereditary deafness. Gap junctions formed by different connexins have unique selectivity to large molecules, so compensating for the loss of one isoform can be challenging in the case of disease causing mutations. We compared the properties of Cx26 mutants T8M and N206S with wild-type channels in transfected cells using dual whole cell voltage clamp and dye flux experiments. Wild-type and mutant channels demonstrated comparable ionic coupling, and their average unitary conductance was ~106 and ~60 pS in 120 mM K
+ -aspartate- and TEA+ -aspartate- solution, respectively, documenting their equivalent permeability to K+ and TEA+ . Comparison of cAMP, Lucifer Yellow (LY), and ethidium bromide (EtBr) transfer revealed differences in selectivity for larger anionic and cationic tracers. cAMP and LY permeability to wild-type and mutant channels was similar, whereas the transfer of EtBr through mutant channels was greatly reduced compared with wild-type junctions. Altered permeability of Cx26 to large cationic molecules suggests an essential role for biochemical coupling in cochlear homeostasis. [ABSTRACT FROM AUTHOR]- Published
- 2008
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10. Human connexin26 and connexin3O form functional heteromeric and heterotypic channels.
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Yum, Sabrina W., Junxian Zhang, Valiunas, Virginijus, Kanaporis, Giedrius, Brink, Peter R., White, Thomas W., and Scherer, Steven S.
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CONNEXINS ,MEMBRANE proteins ,GENETIC mutation ,PROTEINS ,DEAFNESS - Abstract
Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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11. Biophysical characterization of zebrafish connexin35 hemichannels.
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Valiunas, Virginijus, Mui, Riekie, McLachlan, Elizabeth, Valdimarsson, Gunnar, Brink, Peter R., and White, Thomas W.
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CONNEXINS ,ZEBRA danio ,MEMBRANE proteins ,GAP junctions (Cell biology) ,CELL physiology ,CYTOLOGY - Abstract
A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the C×35/C×36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that C×35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell- attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the C×35 hemichannels (I
hc ) that were activated and increased by stepwise depolarization of membrane potential (Vm) and deactivated by hyper- polarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense C×38. However, water-injected oocytes that were not treated with antisense showed activities attributable to C×38 hemichannels that were easily distinguishable from C×35 hemichannels by a significantly larger unitary conductance (γhc : 250-320 pS). The γhc of C×35 hemichannels exhibited a pronounced Vm dependence; i.e., γhc increased/decreased with relative hyperpolarization/depolarization (γhc was 72 pS at Vm = — 100 mV and 35 pS at Vm = 100 mV). Extrapolation to Vm = 0 mV predicted a γhc of 48 pS, suggesting a unitary conductance of intact C×35 gap junction channels of ∼24 pS. Channel gating was also Vm dependent: open time declined with negative Vm and increased with positive Vm. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo. [ABSTRACT FROM AUTHOR]- Published
- 2004
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12. Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin.
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Christie, Tara L., Mui, Rickie, White, Thomas W., and Valdimarsson, Gunnar
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ZEBRA danio ,MOLECULAR cloning ,FUNCTIONAL analysis ,CONNEXINS ,GAP junctions (Cell biology) ,MESSENGER RNA - Abstract
In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins. the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (z fCx45.6), which exhibits 53% amino acid identity to chick Cxd2. was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf. after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4. zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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13. Connexin disorders of the ear, skin, and lens
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Gerido, Dwan A. and White, Thomas W.
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CONNEXINS , *MEMBRANE proteins , *DISEASES , *SKIN - Abstract
Gap junctions provide coupled cells with a direct pathway for sharing ions, nutrients, and small metabolites, thus helping to maintain homeostasis in various tissues. Abnormal function and/or expression of specific connexin genes has been linked to several diseases, including genetic deafness, skin disease, peripheral neuropathies, and cataracts. Research has provided significant insight into the function of gap junction proteins in both in vitro and in vivo models; however, questions regarding the exact mechanisms by which connexin related diseases occur in mammalian systems remain. Here, we discuss the disease states that are related to three human connexin genes, Cx26 (GJB2), Cx46 (GJA3) and Cx50 (GJA8), and recent scientific evidence characterizing those diseases in various experimental models. [Copyright &y& Elsevier]
- Published
- 2004
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14. Virtual cloning, functional expression, and gating analysis of human connexin31.9.
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White, Thomas W., Srinivas, Miduturu, Ripps, Harris, Trovato-Salinaro, Angela, Condorelli, Daniele F., and Bruzzone, Roberto
- Subjects
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CONNEXINS , *GENETIC code , *ELECTRIC conductivity - Abstract
Identifies a novel connexin sequence in the human Expressed Sequence Tagged database that encodes connexin31.9. Sequence analysis and tissue distribution; Voltage-gating behavior of connexin31.9; Pharmacological inhibition of connexin31.9; Unitary conductance of connexin31.9 channels.
- Published
- 2002
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15. GENETIC DISEASES AND GENE KNOCKOUTS REVEAL DIVERSE CONNEXIN FUNCTIONS.
- Author
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White, Thomas W. and Paul, David L.
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CONNEXINS , *BIOLOGY , *GAP junctions (Cell biology) - Abstract
Focuses on the functions of vertebrate connexins as characterized by genetic diseases and gene knockouts. Information on the vertebrate intercellular channels; Details on the invertebrate gap junction channles; Mutations of connexin in human diseases.
- Published
- 1999
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16. Functional characteristics of skate connexin35, a member of the γ subfamily of connexins expressed in the vertebrate retina.
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White, Thomas W., Deans, Michael R., O'Brien, John, Al‐Ubaidi, Muayyad R., Goodenough, Daniel A., Ripps, Harris, and Bruzzone, Roberto
- Subjects
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CONNEXINS , *RETINA , *MEMBRANE proteins , *GAP junctions (Cell biology) - Abstract
Abstract Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the γ subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage-gated at transjunctional potentials ≥ 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the α or β subfamilies. Voltage-activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 μm quinine resulted in a 1.8-fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage-activated currents of Cx46 hemichannels were also enhanced 1.6-fold following quinine treatment, whereas Cx43-injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells. [ABSTRACT FROM AUTHOR]
- Published
- 1999
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17. The cellular internet: On-line with connexins.
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Bruzzone, Roberto and White, Thomas W.
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CONNEXINS , *CELL communication - Abstract
Discusses the importance of connexins in communication between cells. Structure of intercellular channels; Connexin proteins and genes; Responsibilities of connexins; Languages of connexins; Lessons from knockout mice and human diseases.
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- 1996
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18. Connections with connexins: the molecular basis of direct intercellular signaling.
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Bruzzone, Robeto, White, Thomas W., and Paul, David L.
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CONNEXINS , *CELL communication , *GAP junctions (Cell biology) , *GENES , *GENETIC mutation , *MOLECULAR biology , *HOMEOSTASIS - Abstract
Adjacent cells share ions, second messengers and small metabolites through intercellular channels which are present in gap junctions. This type of intercellular communication permits coordinated cellular activity, a critical feature for organ homeostasis during development and adult life of multicellular organisms. Intercellular channels are structurally more complex than other ion channels, because a complete cell-to-cell channel spans two plasma membranes and results from the association of two half channels, or connexons, contributed separately by each of the two participating cells. Each connexon, in turn, is a multimeric assembly of protein subunits. The structural proteins comprising these channels, collectively called connexins, are members of a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeability of intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter-connexin interactions and uncovered novel characteristics of the channel permeability and gating behavior. Structure/function studies have begun to provide a molecular understanding of the significance of connexin diversity and demonstrated the unique regulation of connexins by tyrosine kinases and oncogenes. Finally, mutations in two connexin genes have been linked to human diseases. The development of more specific approaches (dominant negative mutants, knockouts, transgenes) to study the functional role of connexins in organ homeostasis is providing a new perception about the significance of connexin diversity and the regulation of intercellular communication. [ABSTRACT FROM AUTHOR]
- Published
- 1996
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19. Is half of a lens gap junction channel better than none? Focus on "Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46".
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White, Thomas W.
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CATARACT , *CONNEXIN genetics , *CONNEXINS , *GENETIC mutation , *GENETICS , *PHYSIOLOGY ,GENETICS of eye abnormalities - Abstract
The article discusses the functional properties of two cataract-associated mutations connexins 46 that is located in its amino-terminal and also discusses defects in gap functional coupling. He reports that mutations in lens connexins are the major cause of human hereditary vision loss which results in nonsyndromic hereditary cataract. He mentions that cataract-causing mutations in connexins-46 are due to connexin hemichannels.
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- 2013
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20. Human diseases associated with connexin mutations.
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Srinivas, Miduturu, Verselis, Vytas K., and White, Thomas W.
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GAP junctions (Cell biology) , *CONNEXINS , *GENETIC disorders , *PATHOLOGICAL physiology , *GENETIC mutation - Abstract
Gap junctions and hemichannels comprised of connexins impact many cellular processes. Significant advances in our understanding of the functional role of these channels have been made by the identification of a host of genetic diseases caused by connexin mutations. Prominent features of connexin disorders are the inability of other connexins expressed in the same cell type to compensate for the mutated one, and the ability of connexin mutants to dominantly influence the activity of other wild-type connexins. Functional studies have begun to identify some of the underlying mechanisms whereby connexin channel mutation contributes to the disease state. Detailed mechanistic understanding of these functional differences will help to facilitate new pathophysiology driven therapies for the diverse array of connexin genetic disorders. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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21. The cataract-inducing S50P mutation in Cx50 dominantly alters the channel gating of wild-type lens connexins.
- Author
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DeRosa, Adam M., Chun-Hong Xia, Xiaohua Gong, and White, Thomas W.
- Subjects
GENETIC mutation ,CATARACT ,CONNEXINS ,HELA cells ,GAP junctions (Cell biology) - Abstract
Mutations within connexin50 (Cx50) have been linked to various cataract phenotypes. To determine the mechanism behind cataract formation we used the paired Xenopus oocyte system in conjunction with transfected HeLa cells and genetically engineered mouse models to examine the functional characteristics of gap junctions in which a cataract-causing mutant of Cx50 (hereafter referred to as Cx50-S50P) is expressed. Channels comprising Cx50-S50P subunits alone failed to induce electrical coupling. However, the mixed expression of Cx50-S50P and wild-type subunits of either Cx50 or Cx46 -- to create heteromeric gap junctions -- resulted in functional intercellular channels with altered voltage-gating properties compared with homotypic wild-type channels. Additionally, immunofluorescence microscopy showed that channels of Cx50-S50P subunits alone failed to localize to the plasma membrane -- unlike channels composed of Cx46 subunits, which concentrated at cell-cell appositions. Cx50-S50P colocalized with wild-type Cx46 in both transfected HeLa cells in vitro and mouse lens sections in vivo. Taken together, these data define the electrophysiological properties and intracellular targeting of gap junctions formed by the heteromeric combination of Cx50 or Cx46 and Cx50-S50P mutant proteins. Additionally, mixed channels displayed significantly altered gating properties, a phenomenon that may contribute to the cataract that is associated with this mutation. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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22. Knock-in of α3 connexin prevents severe cataracts caused by an α8 point mutation.
- Author
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Chun-hong Xia, Cheung, Debra, DeRosa, Adam M., Bo Chang, Woo-Kuen Lo, White, Thomas W., and Xiaohua Gong
- Subjects
CONNEXINS ,CATARACT ,MEMBRANE proteins ,GENE expression ,HOMEOSTASIS ,PHYSIOLOGICAL control systems - Abstract
A G22R point mutation in α8 connexin (Cx50) has been previously shown to cause a severe cataract by interacting with endogenous wild-type α3 connexin (Cx46) in mouse lenses. Here, we tested whether a knocked-in α3 connexin expressed on the locus of the endogenous α8 connexin could modulate the severe cataract caused by the α8-G22R mutation. We found that the α3(-/-)α8(G22R/-) mice developed severe cataracts with disrupted inner fibers and posterior rupture while the α3(-/-)α8(G22R/KIα3) lens contained relatively normal inner fibers without lens posterior rupture. The α8-G22R mutant proteins produced typical punctate staining of gap junctions between fiber cells of α3(-/-)α8(G22R/KIα3) lenses, but not in those of α3(-/-) α8(G22R/-) lenses. Thus, we hypothesize that the knocked-in α3 connexin subunits interact with the α8-G22R connexin subunits to form functional gap junction channels and rescue the lens phenotype. Using an electrical coupling assay consisting of paired Xenopus oocytes, we demonstrated that only co-expression of mutant α8-G22R and wild-type α3 connexin subunits forms functional gap junction channels with reduced conductance and altered voltage sensitivity compared with the channels formed by α3 connexin subunits alone. Thus, knocked-in α3 connexin and mutant α8-G22R connexin probably form heteromeric gap junction channels that influence lens homeostasis and lens transparency. [ABSTRACT FROM AUTHOR]
- Published
- 2006
23. Connexin Mutations Causing Skin Disease and Deafness Increase Hemichannel Activity and Cell Death when Expressed in Xenopus Oocytes.
- Author
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Lee, Jack R., DeRosa, Adam M., and White, Thomas W.
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CONNEXINS , *SKIN diseases , *DEAFNESS , *GENETIC mutation , *GAP junctions (Cell biology) , *XENOPUS , *CELL death - Abstract
Mutations in the GJB2 gene-encoding connexin 26 (Cx26) have been linked to skin disorders and genetic deafness. However, the severity and type of the skin disorders caused by Cx26 mutations are heterogeneous. Here we explored the effect of Cx26 KID syndrome-associated mutations, G12R, S17F, and D50N on channel function. The Cx26 N14K mutation was also examined that is associated with deafness but has a skin disorder distinct from the KID syndrome mutations. The proteins were all expressed in Xenopus oocytes with levels equal to wild-type Cx26. The G12R, N14K, and D50N mutations resulted in larger hemichannel currents than the wild-type-expressing cells, but the S17F mutation resulted in a complete loss of hemichannel activity. Elevated hemichannel activity correlated with an increased cell death. This result could be reversed through the elevation of calcium (Ca2+) in the extracellular media. Functional gap junctions were only produced by paired N14K cells, which had a similar conductance level to wild type, even though they exhibited a complete loss of voltage sensitivity. This set of data confirms that aberrant hemichannel activity is a common feature of Cx26 mutations associated with KID syndrome, and this may contribute to a loss of cell viability and tissue integrity.Journal of Investigative Dermatology (2009) 129, 870–878; doi:10.1038/jid.2008.335; published online 6 November 2008 [ABSTRACT FROM AUTHOR]
- Published
- 2009
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24. Gap Junctions: Basic Structure and Function.
- Author
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Meşe, Gülistan, Richard, Gabriele, and White, Thomas W.
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GAP junctions (Cell biology) , *CELL junctions , *CONNEXINS , *CELLULAR control mechanisms , *MEMBRANE proteins , *CELL communication , *SKIN diseases - Abstract
Gap junctions allow the exchange of ions, second messengers, and small metabolites between adjacent cells and are formed by two unrelated protein families, the pannexins and connexins. Mutations in connexin genes cause a variety of genetic disorders, implicating a critical role in tissue homeostasis. Association of congenital skin disorders to mutations in different connexins has underscored the importance of gap junctional communication in the skin and its appendages. Here, we discuss the basic structure of gap junction channels and the function of connexin genes that have been associated with human disorders to explore the physiology of intercellular communication in skin.Journal of Investigative Dermatology (2007) 127, 2516–2524; doi:10.1038/sj.jid.5700770 [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
25. Connexin channels in congenital skin disorders.
- Author
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Lilly, Evelyn, Sellitto, Caterina, Milstone, Leonard M., and White, Thomas W.
- Subjects
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CONNEXINS , *SKIN disease treatment , *GAP junctions (Cell biology) , *CELL differentiation , *GENETIC mutation - Abstract
Gap junctions and hemichannels comprised of connexins influence epidermal proliferation and differentiation. Significant advances in our understanding of the functional role of connexins in the skin have been made by studying the diseases caused by connexin mutations. Eleven clinically defined cutaneous disorders with an overlapping spectrum of phenotypes are caused by mutations in five different connexin genes, highlighting that disease presentation must be deciphered with an understanding of how connexin functions are affected. Increasing evidence suggests that the skin diseases produced by connexin mutations result from dominant gains of function. In palmoplantar keratoderma with deafness, the connexin 26 mutations transdominantly alter the function of wild-type connexin 43 and create leaky heteromeric hemichannels. In keratitis–ichthyosis–deafness syndrome, different connexin 26 mutations can either form dominant hemichannels with altered calcium regulation or increased calcium permeability, leading to clinical subtypes of this syndrome. It is only with detailed understanding of these subtle functional differences that we can hope to create successful pathophysiology driven therapies for the connexin skin disorders. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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26. Connexin Controls Cell-Cycle Exit and Cell Differentiation by Directly Promoting Cytosolic Localization and Degradation of E3 Ligase Skp2.
- Author
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Shi, Qian, Gu, Sumin, Yu, X. Sean, White, Thomas W., Banks, Eric A., and Jiang, Jean X.
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CELL cycle proteins , *CELL differentiation , *CYTOSOL , *LIGASES , *CONNEXINS , *PHYSIOLOGY - Abstract
Summary Connexins and connexin channels play important roles in cell growth/differentiation and tumorigenesis. Here, we identified a relationship between a connexin molecule and a critical cell-cycle regulator. Our data show that connexin (Cx) 50 regulated lens cell-cycle progression and differentiation by modulating expression of cyclin-dependent kinase inhibitor p27/p57 and E3 ubiquitin ligase Skp2. Cx50 directly interacted with and retained Skp2 in the cytosol by masking the nuclear targeting domain of Skp2, and this effect was supported by an increased nuclear localization of Skp2, disruption of Skp2 interaction with importin-7, and decreased levels of p27/p57 in mouse lenses lacking Cx50. As a result, Cx50 increased auto-ubiquitination and subsequent degradation of Skp2. A mutation (V362E) on the C terminus of Cx50 disrupted the interaction between Cx50 and Skp2 and completely abolished such effects. Therefore, this study identifies a role for connexins in regulating cell-cycle modulators and, consequently, cell growth and differentiation. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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27. Aberrant Connexin26 Hemichannels Underlying Keratitis-Ichthyosis-Deafness Syndrome Are Potently Inhibited by Mefloquine.
- Author
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Levit, Noah A, Sellitto, Caterina, Wang, Hong-Zhan, Li, Leping, Srinivas, Miduturu, Brink, Peter R, and White, Thomas W
- Subjects
- *
KERATITIS , *ICHTHYOSIS , *MEFLOQUINE , *CONNEXINS , *GAP junctions (Cell biology) , *ECTODERMAL dysplasia , *THERAPEUTICS - Abstract
Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes nonsyndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small-molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine (MFQ) inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50∼16 μM), using an extracellular divalent cation, zinc (Zn++), as a nonspecific positive control for comparison (IC50∼3 μM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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28. Connexin 46 (Cx46) Gap Junctions Provide a Pathway for the Delivery of Glutathione to the Lens Nucleus.
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Slavi, Nefeli, Rubinos, Clio, Leping Li, Sellitto, Caterina, White, Thomas W., Mathias, Richard, and Srinivas, Miduturu
- Subjects
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GLUTATHIONE , *CELL nuclei , *GAP junctions (Cell biology) , *CONNEXINS , *CELLULAR signal transduction - Abstract
Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (PNa/PCl ~5 for Cx46 and ~3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (PNa/PGSH ~12 for Cx46 and ~8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
29. Pathological hemichannels associated with human Cx26 mutations causing Keratitis–Ichthyosis–Deafness syndrome
- Author
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Levit, Noah A., Mese, Gulistan, Basaly, Mena-George R., and White, Thomas W.
- Subjects
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GENETIC mutation , *KERATITIS , *ICHTHYOSIS , *CONNEXINS , *GAP junctions (Cell biology) , *CYTOPLASM - Abstract
Abstract: Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis–Ichthyosis–Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
30. The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium
- Author
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DeRosa, Adam M., Meşe, Gülistan, Li, Leping, Sellitto, Caterina, Brink, Peter R., Gong, Xiaohua, and White, Thomas W.
- Subjects
- *
CATARACT , *CELL communication , *GENETIC mutation , *LABORATORY mice , *CONNEXINS , *PATHOLOGICAL physiology - Abstract
Abstract: Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling ≥90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell–cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell–cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation. [Copyright &y& Elsevier]
- Published
- 2009
- Full Text
- View/download PDF
31. Gap Junction Channels Exhibit Connexin-specific Permeability to Cyclic Nucleotides.
- Author
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Kanaporis, Giedrius, Mese, Gulistan, Valiuniene, Laima, White, Thomas W., Brink, Peter R., and Valiunas, Virginijus
- Subjects
- *
GAP junctions (Cell biology) , *CONNEXINS , *CYCLIC nucleotides , *CELLS , *BIOCHEMISTRY - Abstract
Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance arid intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (Ih) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in Ih, current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K+ permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were ∼10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 ≥ Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
32. Connections Between Connexins, Calcium, and Cataracts in the Lens.
- Author
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Gao, Junyuan, Sun, Xiurong, Martinez-Wittinghan, Francisco J., Gong, Xiaohua, White, Thomas W., and Mathias, Richard T.
- Subjects
- *
CRYSTALLINE lens , *CONNEXINS , *CALCIUM , *CATARACT , *GAP junctions (Cell biology) , *PHYSIOLOGY - Abstract
There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was ∼0.5 S/cm² of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was ∼1.0 S/cm² and calcium varied from ∼500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to ∼2 µM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of Cx46 causes loss of coupling of mature fiber cells; the efflux path for calcium is therefore blocked; calcium accumulates in the central cells; at concentrations above 1 µM (from the center to about half way out of a 3-wk-old lens) Lp82 is activated; Lp82 cleaves cytoplasmic proteins (crystallins) in central cells; and the cleaved proteins aggregate and scatter light. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
33. Connexin 48.5 Is Required for Normal Cardiovascular Function and Lens Development in Zebrafish Embryos.
- Author
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Shaohong Cheng, Shakespeare, Teresa, Mui, Rickie, White, Thomas W., and Valdimarsson, Gunnar
- Subjects
- *
CONNEXINS , *CARDIOVASCULAR system , *ZEBRA danio , *XENOPUS , *CELL communication , *HOMEOSTASIS , *MAMMALS - Abstract
Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
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