Eric C Beyer | University of Chicago (original) (raw)

Papers by Eric C Beyer

Research paper thumbnail of Editorial: Ion channels, pumps, and transporters in lens physiology and disease

Frontiers in Physiology, Nov 3, 2022

Research paper thumbnail of Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle

Journal of Biological Chemistry, 1980

Research paper thumbnail of The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation

Investigative Opthalmology & Visual Science, 2019

PURPOSE. Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expres... more PURPOSE. Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation. METHODS. Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca 2þ ] i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images. RESULTS. In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wildtype at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca 2þ ] i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts. CONCLUSIONS. Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

Research paper thumbnail of Connexin46 mutations linked to congenital cataract show loss of gap junction channel function

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....

Research paper thumbnail of Lens Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx 46 fs 380 Mice

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.

Research paper thumbnail of Mechanisms for the coordination of intercellular calcium signaling in insulin-secreting cells

Journal of Cell Science, 1997

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...

Research paper thumbnail of Do Connexin Mutants Cause Cataracts by Perturbing Glutathione Levels and Redox Metabolism in the Lens?

Biomolecules, 2020

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...

Research paper thumbnail of Peptide inhibitors of intercellular communication

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2000

Research paper thumbnail of Disruption of the lens circulation causes calcium accumulation and precipitates in connexin mutant mice

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...

Research paper thumbnail of Faculty of 1000 evaluation for Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome

F1000 - Post-publication peer review of the biomedical literature, 2017

Background: Aberrantly functioning Cx26 hemichannels are a common feature of GJB2 mutations causi... more Background: Aberrantly functioning Cx26 hemichannels are a common feature of GJB2 mutations causing syndromic deafness. Results: pH and Zn 2ϩ , factors that inhibit hemichannels, are less effective in the A40V KID syndrome mutant. Conclusion: Impaired inhibition by pH and Zn 2ϩ can contribute to the pathogenesis of KID syndrome. Significance: Data provide new insights into Cx26 hemichannel function and possible contributions to tissue function. Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca 2؉. This study presents two new possible contributing factors, pH and Zn 2؉. Plasma pH levels and micromolar concentrations of Zn 2؉ inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H ؉ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn 2؉. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn 2؉ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn 2؉. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism. Mutations in the GJB2 gene that codes for the connexin 26 (Cx26) gap junction (GJ) 2 protein result in non-syndromic and

Research paper thumbnail of Faculty of 1000 evaluation for Computational Studies of Molecular Permeation through Connexin26 Channels

F1000 - Post-publication peer review of the biomedical literature, 2017

A signal property of connexin channels is the ability to mediate selective diffusive movement of ... more A signal property of connexin channels is the ability to mediate selective diffusive movement of molecules through plasma membrane(s), but the energetics and determinants of molecular movement through these channels have yet to be understood. Different connexin channels have distinct molecular selectivities that cannot be explained simply on the basis of size or charge of the permeants. To gain insight into the forces and interactions that underlie selective molecular permeation, we investigated the energetics of two uncharged derivatized sugars, one permeable and one impermeable, through a validated connexin26 (Cx26) channel structural model, using molecular dynamics and associated analytic tools. The system is a Cx26 channel equilibrated in explicit membrane/solvent, shown by Brownian dynamics to reproduce key conductance characteristics of the native channel. The results are consistent with the known difference in permeability to each molecule. The energetic barriers extend through most of the pore length, rather than being highly localized as in ion-specific channels. There is little evidence for binding within the pore. Force decomposition reveals how, for each tested molecule, interactions with water and the Cx26 protein vary over the length of the pore and reveals a significant contribution from hydrogen bonding and interaction with K þ. The flexibility of the pore width varies along its length, and the tested molecules have differential effects on pore width as they pass through. Potential sites of interaction within the pore are defined for each molecule. The results suggest that for the tested molecules, differences in hydrogen bonding and entropic factors arising from permeant flexibility substantially contribute to the energetics of permeation. This work highlights factors involved in selective molecular permeation that differ from those that define selectivity among atomic ions.

Research paper thumbnail of Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx46fs380 Mice

Investigative Opthalmology & Visual Science, 2017

PURPOSE. Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexi... more 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. METHODS. Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses. RESULTS. Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses. CONCLUSIONS. Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

Research paper thumbnail of Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract

PLOS ONE, 2017

Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnorm... more Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis. Methods We performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells. Results Exome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells.

Research paper thumbnail of Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

Molecular and Cellular Biology, 1990

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...

Research paper thumbnail of Connexin23 deletion does not affect lens transparency

Experimental Eye Research, 2016

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and g... more While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the coexpressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.

Research paper thumbnail of Expression of the Gap junction connexin43 in embryonic chick lens : molecular cloning, ultrastructural localization, and post-translational phosphorylation

The Journal of Membrane Biology, 1990

Research paper thumbnail of Method of modulating angiogenesis

Research paper thumbnail of The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses

Journal of Biological Chemistry, 2016

Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation.... more Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive ␣B-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo. Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1␣ or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2␣ (2.4-and 3.3fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9-and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.

Research paper thumbnail of Mutagenesis Of Charged Residues In The N-terminal alpha-helix Of Connexin37 Reveals An Essential Lysine Residue

Research paper thumbnail of Regulation of Lens Gap Junctions

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.

Research paper thumbnail of Editorial: Ion channels, pumps, and transporters in lens physiology and disease

Frontiers in Physiology, Nov 3, 2022

Research paper thumbnail of Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle

Journal of Biological Chemistry, 1980

Research paper thumbnail of The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation

Investigative Opthalmology & Visual Science, 2019

PURPOSE. Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expres... more PURPOSE. Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation. METHODS. Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca 2þ ] i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images. RESULTS. In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wildtype at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca 2þ ] i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts. CONCLUSIONS. Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

Research paper thumbnail of Connexin46 mutations linked to congenital cataract show loss of gap junction channel function

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....

Research paper thumbnail of Lens Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx 46 fs 380 Mice

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.

Research paper thumbnail of Mechanisms for the coordination of intercellular calcium signaling in insulin-secreting cells

Journal of Cell Science, 1997

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...

Research paper thumbnail of Do Connexin Mutants Cause Cataracts by Perturbing Glutathione Levels and Redox Metabolism in the Lens?

Biomolecules, 2020

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...

Research paper thumbnail of Peptide inhibitors of intercellular communication

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2000

Research paper thumbnail of Disruption of the lens circulation causes calcium accumulation and precipitates in connexin mutant mice

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...

Research paper thumbnail of Faculty of 1000 evaluation for Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome

F1000 - Post-publication peer review of the biomedical literature, 2017

Background: Aberrantly functioning Cx26 hemichannels are a common feature of GJB2 mutations causi... more Background: Aberrantly functioning Cx26 hemichannels are a common feature of GJB2 mutations causing syndromic deafness. Results: pH and Zn 2ϩ , factors that inhibit hemichannels, are less effective in the A40V KID syndrome mutant. Conclusion: Impaired inhibition by pH and Zn 2ϩ can contribute to the pathogenesis of KID syndrome. Significance: Data provide new insights into Cx26 hemichannel function and possible contributions to tissue function. Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca 2؉. This study presents two new possible contributing factors, pH and Zn 2؉. Plasma pH levels and micromolar concentrations of Zn 2؉ inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H ؉ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn 2؉. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn 2؉ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn 2؉. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism. Mutations in the GJB2 gene that codes for the connexin 26 (Cx26) gap junction (GJ) 2 protein result in non-syndromic and

Research paper thumbnail of Faculty of 1000 evaluation for Computational Studies of Molecular Permeation through Connexin26 Channels

F1000 - Post-publication peer review of the biomedical literature, 2017

A signal property of connexin channels is the ability to mediate selective diffusive movement of ... more A signal property of connexin channels is the ability to mediate selective diffusive movement of molecules through plasma membrane(s), but the energetics and determinants of molecular movement through these channels have yet to be understood. Different connexin channels have distinct molecular selectivities that cannot be explained simply on the basis of size or charge of the permeants. To gain insight into the forces and interactions that underlie selective molecular permeation, we investigated the energetics of two uncharged derivatized sugars, one permeable and one impermeable, through a validated connexin26 (Cx26) channel structural model, using molecular dynamics and associated analytic tools. The system is a Cx26 channel equilibrated in explicit membrane/solvent, shown by Brownian dynamics to reproduce key conductance characteristics of the native channel. The results are consistent with the known difference in permeability to each molecule. The energetic barriers extend through most of the pore length, rather than being highly localized as in ion-specific channels. There is little evidence for binding within the pore. Force decomposition reveals how, for each tested molecule, interactions with water and the Cx26 protein vary over the length of the pore and reveals a significant contribution from hydrogen bonding and interaction with K þ. The flexibility of the pore width varies along its length, and the tested molecules have differential effects on pore width as they pass through. Potential sites of interaction within the pore are defined for each molecule. The results suggest that for the tested molecules, differences in hydrogen bonding and entropic factors arising from permeant flexibility substantially contribute to the energetics of permeation. This work highlights factors involved in selective molecular permeation that differ from those that define selectivity among atomic ions.

Research paper thumbnail of Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx46fs380 Mice

Investigative Opthalmology & Visual Science, 2017

PURPOSE. Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexi... more 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. METHODS. Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses. RESULTS. Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses. CONCLUSIONS. Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

Research paper thumbnail of Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract

PLOS ONE, 2017

Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnorm... more Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis. Methods We performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells. Results Exome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells.

Research paper thumbnail of Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

Molecular and Cellular Biology, 1990

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...

Research paper thumbnail of Connexin23 deletion does not affect lens transparency

Experimental Eye Research, 2016

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and g... more While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the coexpressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.

Research paper thumbnail of Expression of the Gap junction connexin43 in embryonic chick lens : molecular cloning, ultrastructural localization, and post-translational phosphorylation

The Journal of Membrane Biology, 1990

Research paper thumbnail of Method of modulating angiogenesis

Research paper thumbnail of The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses

Journal of Biological Chemistry, 2016

Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation.... more Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive ␣B-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo. Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1␣ or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2␣ (2.4-and 3.3fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9-and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.

Research paper thumbnail of Mutagenesis Of Charged Residues In The N-terminal alpha-helix Of Connexin37 Reveals An Essential Lysine Residue

Research paper thumbnail of Regulation of Lens Gap Junctions

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.