American Journal of Physiology-Cell Physiology, 2000
Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appea... more Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels....
Citation: Minogue PJ, Gao J, Zoltoski RK, et al. Physiological and optical alterations precede th... more Citation: Minogue PJ, Gao J, Zoltoski RK, et al. Physiological and optical alterations precede the appearance of cataracts in Cx46fs380 mice. Invest Ophthalmol Vis Sci. 2017;58:4366– 4374. DOI:10.1167/iovs.17-21684 PURPOSE. Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ~2 months in homozygotes and at ‡4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts.
Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic ... more Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves th...
Cataracts of many different etiologies are associated with oxidation of lens components. The lens... more Cataracts of many different etiologies are associated with oxidation of lens components. The lens is protected by maintenance of a pool of reduced glutathione (GSH) and other antioxidants. Because gap junction channels made of the lens connexins, Cx46 and Cx50, are permeable to GSH, we tested whether mice expressing two different mutants, Cx46fs380 and Cx50D47A, cause cataracts by impairing lens glutathione metabolism and facilitating oxidative damage. Levels of GSH were not reduced in homogenates of whole mutant lenses. Oxidized glutathione (GSSG) and the GSSG/GSH ratio were increased in whole lenses of Cx50D47A, but not Cx46fs380 mice. The GSSG/GSH ratio was increased in the lens nucleus (but not cortex) of Cx46fs380 mice at 4.5 months of age, but it was not altered in younger animals. Carbonylated proteins were increased in Cx50D47A, but not Cx46fs380 lenses. Thus, both mouse lines have oxidizing lens environments, but oxidative modification is greater in Cx50D47A than in Cx46fs3...
American journal of physiology. Cell physiology, 2018
The lens is an avascular organ whose function and survival depend on an internal circulation syst... more The lens is an avascular organ whose function and survival depend on an internal circulation system. Cx46fs380 mice model a human autosomal dominant cataract caused by a mutant lens connexin. In these mice, fiber cell connexin levels and gap junction coupling are severely decreased. The present studies were conducted to examine components of the lens circulation system that might be altered and contribute to the pathogenesis of cataracts. Lenses from wild-type mice and Cx46fs380 heterozygotes and homozygotes were studied at 2 months of age. Cx46fs380-expressing lens fiber cells were depolarized. Cx46fs380 lenses had increased intracellular hydrostatic pressure and concentrations of Na and Ca. The activity of epithelial Na-K-ATPase was decreased in Cx46fs380 lenses. All of these changes were more severe in homozygous than in heterozygous Cx46fs380 lenses. Cx46fs380 cataracts were stained by Alizarin red, a dye used to detect insoluble Ca. These data suggest that the lens internal cir...
Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-w... more Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47...
From Ion Channels to Cell-to-Cell Conversations, 1997
Gap junctions are membrane specializations that contain groups of intercellular channels. These c... more Gap junctions are membrane specializations that contain groups of intercellular channels. These channels are permeable to ions and small molecules up to 1,000 Da. They communicate the cytoplasm of adjacent cells, thereby providing a direct pathway for the passage of ions, nutrients, metabolites and second messengers between coupled cells. Gap junctional channels are made of oligomeric assemblies of members of a family of related proteins called connexins (Cx). These proteins cross the plasma membrane four times leaving the amino and carboxyl termini on the cytoplasmic side (reviewed by Beyer)1.
American Journal of Physiology-Cell Physiology, 2000
Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appea... more Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels....
Citation: Minogue PJ, Gao J, Zoltoski RK, et al. Physiological and optical alterations precede th... more Citation: Minogue PJ, Gao J, Zoltoski RK, et al. Physiological and optical alterations precede the appearance of cataracts in Cx46fs380 mice. Invest Ophthalmol Vis Sci. 2017;58:4366– 4374. DOI:10.1167/iovs.17-21684 PURPOSE. Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ~2 months in homozygotes and at ‡4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts.
Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic ... more Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves th...
Cataracts of many different etiologies are associated with oxidation of lens components. The lens... more Cataracts of many different etiologies are associated with oxidation of lens components. The lens is protected by maintenance of a pool of reduced glutathione (GSH) and other antioxidants. Because gap junction channels made of the lens connexins, Cx46 and Cx50, are permeable to GSH, we tested whether mice expressing two different mutants, Cx46fs380 and Cx50D47A, cause cataracts by impairing lens glutathione metabolism and facilitating oxidative damage. Levels of GSH were not reduced in homogenates of whole mutant lenses. Oxidized glutathione (GSSG) and the GSSG/GSH ratio were increased in whole lenses of Cx50D47A, but not Cx46fs380 mice. The GSSG/GSH ratio was increased in the lens nucleus (but not cortex) of Cx46fs380 mice at 4.5 months of age, but it was not altered in younger animals. Carbonylated proteins were increased in Cx50D47A, but not Cx46fs380 lenses. Thus, both mouse lines have oxidizing lens environments, but oxidative modification is greater in Cx50D47A than in Cx46fs3...
American journal of physiology. Cell physiology, 2018
The lens is an avascular organ whose function and survival depend on an internal circulation syst... more The lens is an avascular organ whose function and survival depend on an internal circulation system. Cx46fs380 mice model a human autosomal dominant cataract caused by a mutant lens connexin. In these mice, fiber cell connexin levels and gap junction coupling are severely decreased. The present studies were conducted to examine components of the lens circulation system that might be altered and contribute to the pathogenesis of cataracts. Lenses from wild-type mice and Cx46fs380 heterozygotes and homozygotes were studied at 2 months of age. Cx46fs380-expressing lens fiber cells were depolarized. Cx46fs380 lenses had increased intracellular hydrostatic pressure and concentrations of Na and Ca. The activity of epithelial Na-K-ATPase was decreased in Cx46fs380 lenses. All of these changes were more severe in homozygous than in heterozygous Cx46fs380 lenses. Cx46fs380 cataracts were stained by Alizarin red, a dye used to detect insoluble Ca. These data suggest that the lens internal cir...
Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-w... more Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47...
From Ion Channels to Cell-to-Cell Conversations, 1997
Gap junctions are membrane specializations that contain groups of intercellular channels. These c... more Gap junctions are membrane specializations that contain groups of intercellular channels. These channels are permeable to ions and small molecules up to 1,000 Da. They communicate the cytoplasm of adjacent cells, thereby providing a direct pathway for the passage of ions, nutrients, metabolites and second messengers between coupled cells. Gap junctional channels are made of oligomeric assemblies of members of a family of related proteins called connexins (Cx). These proteins cross the plasma membrane four times leaving the amino and carboxyl termini on the cytoplasmic side (reviewed by Beyer)1.
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