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Invest Ophthalmol Vis Sci
2019 Sep 03;6012:3821-3829. doi: 10.1167/iovs.19-27302.
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Lens Connexin Channels Have Differential Permeability to the Second Messenger cAMP.
Valiunas V
,
Brink PR
,
White TW
.
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Purpose: Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation.
Methods: We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor 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 were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule.
Results: cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46.
Conclusions: These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.
Figure 1. An electrophysiologic assay for intercellular transfer of cAMP through gap junction channels. (A) When single SpIH expressing cells were whole-cell patched without cAMP in the pipette (cell on left), SpIH currents were small (black line). When cAMP was present in the patch pipette (cell on right), SpIH currents were increased 4- to 5-fold (red line). (B) Cell pairs were co-cultured where one cell expressed the connexin to be tested and SpIH (recipient cell on left, cell 1). The other cell expressed only the connexin to be tested (source cell on right, cell 2). Five hundred micromolar cAMP was delivered via a whole-cell patch pipette into cell 2, and SpIH currents were recorded from cell 1 in the perforated patch mode, before (black line), and after (red line) opening the patch on cell 2.
Figure 2. Cx46, but not Cx50, mediated intercellular transfer of cAMP. (A) Data from a Cx46 cell pair with a gap junctional conductance of 31 nS are shown. The SpIH current recorded from cell 1 over time, in response to voltage pulses of 0 to 100 mV, returning to a tail potential of +50 mV. At time 0, the patch in cell 2 was opened initiating cAMP delivery. The SpIH tail current over time increased approximately 4-fold. (B) Cx50 failed to mediate transfer of cAMP. The SpIH current remained constant in the recipient cell after 500μM cAMP was released in the source cell. This Cx50 cell pair had a gap junctional conductance of 16 nS. (C) Expanded SpIH currents from when the whole cell patch was first opened (black asterisk/trace), and 280 seconds later (red asterisk/trace). The Cx46 cell pair showed an approximately 4-fold increase in SpIH tail currents. For the Cx50 cell pair, the SpIH tail currents were of the same magnitude, indicating little, or no, intercellular cAMP transfer through Cx50 channels. Data from a Cx43 cell pair with a conductance of 6 nS are shown for comparison.
Figure 3. Quantitative comparison of intercellular cAMP transfer between the different lens connexins. (A) The change in the SpIH tail current was plotted versus time for individual cell pairs expressing Cx43 (filled circles), Cx46 (open triangles), or Cx50 (open circles). (B) A plot of the normalized tail current versus time recorded from the recipient cell after cAMP injection into the source cell for the Cx43 and Cx46 expressing cell pair. The SpIH activation time corresponds to the time when SpIH current reaches saturation in the recipient cell. The slopes of first order regressions over the linear part of the plot (dashed lines) were used to calculate the current activation rate (ΔI/Δt). (C) Expanded view of the SpIH tail current data for the Cx50 cell pair shown in (A). In the Cx50 cell pairs where the SpIH current increased, it never reached saturation during the finite period that cells could be recorded from, so ΔI/Δt was estimated from fitting the current increase over the entire range of data available.
Figure 4. Summary quantification of SpIH current and gap junction conductance data. (A) The fold-increase in the SpIH tail current after cAMP delivery to the neighboring cell was plotted for individual cell pairs expressing Cx43 (filled circles, n = 9), Cx46 (open triangles, n = 7), or Cx50 (open circles, n = 10). Mean values of the data are plotted as horizontal lines, with standard deviations plotted as vertical error bars. (B) Plots of the time required for the SpIH tail current to reach saturation in Cx43 and Cx46 cell pairs. For Cx50, the time elapsed without the SpIH tail current reaching saturation was plotted. (C) Plots of the total gap junctional conductance measured between cell pairs expressing Cx43, Cx46, or Cx50.
Figure 5. The SpIH current activation rate is dependent on the magnitude of gap junctional coupling between the cells. The SpIH tail current activation rate (ΔI/Δt) was plotted against gap junctional conductance for Cx43 (filled circles, n = 9), Cx46 (open triangles, n = 7), and Cx50 (open circles, n = 10). The solid lines correspond to first order regressions with the slopes given in the Table.
Figure 6. Visualization of cyclic nucleotide passage through Cx43 and Cx50 gap junction channels using fluorescent ε-cAMP. (A) A pipette filled with 5 mM ε-cAMP was attached to a single cell within a group of HeLa cells expressing Cx43. After opening the patch in whole-cell mode, (B) dye transfer was detected to half of the neighboring cells within 5 minutes, and (C) a majority of the cells within 10 minutes. (D) In Cx50 transfected HeLa cells, (E, F) negligible transfer of ε-cAMP to neighboring cells was observed at 5 or 10 minutes following the opening of the patch pipette.
Figure 7. Quantitative comparison of ε-cAMP flux through Cx43 and Cx50 channels. Simultaneous measurements of gap junction conductance and ε-cAMP flux were made in cell pairs. (A) In a Cx43 expressing cell pair with conductance of 12 nS, (B) high levels of ε-cAMP fluorescence were recorded in the recipient cell 5 minutes after the patch pipette was opened in the donor cell. (C) In a cell pair expressing Cx50 with a conductance of 16 nS, (D) very little passage of ε-cAMP into the recipient cell was seen after 5 minutes. (E) The mean (±SD) conductance for all tested Cx43 and Cx50 cell pairs (n = 3). (F) The ratio of ε-cAMP fluorescent intensity in the recipient cell compared with the donor cell for Cx43 and Cx50.
Abell,
The role of adenosine 3',5'-cyclic monophosphate in the regulation of mammalian cell division.
1973, Pubmed
Abell,
The role of adenosine 3',5'-cyclic monophosphate in the regulation of mammalian cell division.
1973,
Pubmed
Ayad,
Heteromeric, but not homomeric, connexin channels are selectively permeable to inositol phosphates.
2006,
Pubmed
Beltramello,
Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness.
2005,
Pubmed
Berthoud,
Roles and regulation of lens epithelial cell connexins.
2014,
Pubmed
Berthoud,
Loss of function and impaired degradation of a cataract-associated mutant connexin50.
2003,
Pubmed
Boswell,
Cross-talk between fibroblast growth factor and bone morphogenetic proteins regulates gap junction-mediated intercellular communication in lens cells.
2008,
Pubmed
Brewitt,
Periods of oscillatory growth in developing ocular lens correspond with cell cycle times.
1992,
Pubmed
Brewitt,
Growth and transparency in the lens, an epithelial tissue, stimulated by pulses of PDGF.
1988,
Pubmed
Bukauskas,
Gap junction channel gating.
2004,
Pubmed
Bukauskas,
Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells.
1995,
Pubmed
Bukauskas,
Temperature dependence of gap junction properties in neonatal rat heart cells.
1993,
Pubmed
Fan,
Perforated patch recording with beta-escin.
1998,
Pubmed
Foster,
In vivo imaging of embryonic development in the mouse eye by ultrasound biomicroscopy.
2003,
Pubmed
Friedman,
Role of cyclic nucleotides in cell growth and differentiation.
1976,
Pubmed
Gauss,
Molecular identification of a hyperpolarization-activated channel in sea urchin sperm.
1998,
Pubmed
,
Echinobase
Gerido,
Connexin disorders of the ear, skin, and lens.
2004,
Pubmed
Gilula,
Metabolic coupling, ionic coupling and cell contacts.
1972,
Pubmed
Goldberg,
Selective permeability of gap junction channels.
2004,
Pubmed
Gong,
Disruption of alpha3 connexin gene leads to proteolysis and cataractogenesis in mice.
1997,
Pubmed
Goodenough,
Gap junction dynamics and intercellular communication.
1978,
Pubmed
Grimes,
Possible cyclic adenosine monophosphate mediation in isoproterenol-induced suppression of cell division in rat lens epithelium.
1972,
Pubmed
Harris,
Emerging issues of connexin channels: biophysics fills the gap.
2001,
Pubmed
Harris,
Connexin channel permeability to cytoplasmic molecules.
2007,
Pubmed
Hernandez,
Unitary permeability of gap junction channels to second messengers measured by FRET microscopy.
2007,
Pubmed
Hopperstad,
Properties of gap junction channels formed by Cx46 alone and in combination with Cx50.
2000,
Pubmed
Ireland,
Beta-adrenergic mechanisms affect cell division and differentiation in cultured chick lens epithelial cells.
1993,
Pubmed
Jiang,
Gap junctions or hemichannel-dependent and independent roles of connexins in cataractogenesis and lens development.
2010,
Pubmed
Kanaporis,
Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides.
2008,
Pubmed
,
Echinobase
Kanaporis,
Gap junction permeability: selectivity for anionic and cationic probes.
2011,
Pubmed
Kar,
Biological role of connexin intercellular channels and hemichannels.
2012,
Pubmed
Le,
A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens.
2001,
Pubmed
Lovicu,
Cell signaling in lens development.
2006,
Pubmed
Martinez,
Differential regulation of Connexin50 and Connexin46 by PI3K signaling.
2015,
Pubmed
Martinez-Wittinghan,
Dominant cataracts result from incongruous mixing of wild-type lens connexins.
2003,
Pubmed
Mathias,
Lens gap junctions in growth, differentiation, and homeostasis.
2010,
Pubmed
McAvoy,
Lens development.
1999,
Pubmed
McAvoy,
Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration.
1989,
Pubmed
Meşe,
Connexin26 deafness associated mutations show altered permeability to large cationic molecules.
2008,
Pubmed
Mu,
In vivo quantification of embryonic and placental growth during gestation in mice using micro-ultrasound.
2008,
Pubmed
Plum,
Unique and shared functions of different connexins in mice.
2000,
Pubmed
Rackauskas,
Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45.
2007,
Pubmed
Robinson,
An essential role for FGF receptor signaling in lens development.
2006,
Pubmed
Sakai,
The electrical behaviour of rat connexin46 gap junction channels expressed in transfected HeLa cells.
2003,
Pubmed
Schneider,
NIH Image to ImageJ: 25 years of image analysis.
2012,
Pubmed
Secrist,
Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties.
1972,
Pubmed
Secrist,
Fluorescent modification of adenosine 3',5'-monophosphate: spectroscopic properties and activity in enzyme systems.
1972,
Pubmed
Sellitto,
The Phosphoinosotide 3-Kinase Catalytic Subunit p110α is Required for Normal Lens Growth.
2016,
Pubmed
Sellitto,
Connexin50 is essential for normal postnatal lens cell proliferation.
2004,
Pubmed
Shin,
Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate.
2001,
Pubmed
,
Echinobase
Šikić,
A stochastic model of eye lens growth.
2015,
Pubmed
Valiunas,
Cardiac gap junction channels show quantitative differences in selectivity.
2002,
Pubmed
Valiunas,
Electrical properties of gap junction hemichannels identified in transfected HeLa cells.
2000,
Pubmed
Valiunas,
Gap junction channels formed by coexpressed connexin40 and connexin43.
2001,
Pubmed
Valiunas,
Biophysical properties of mouse connexin30 gap junction channels studied in transfected human HeLa cells.
1999,
Pubmed
Veenstra,
Selectivity of connexin-specific gap junctions does not correlate with channel conductance.
1995,
Pubmed
Von Sallmann,
Effects of isoproterenol and cyclic AMP derivatives on cell division in cultured rat lenses.
1974,
Pubmed
White,
Nonredundant gap junction functions.
2003,
Pubmed
White,
Prenatal lens development in connexin43 and connexin50 double knockout mice.
2001,
Pubmed
White,
Intercellular communication in the eye: clarifying the need for connexin diversity.
2000,
Pubmed
White,
Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts.
1998,
Pubmed
White,
Unique and redundant connexin contributions to lens development.
2002,
Pubmed
White,
Optimal lens epithelial cell proliferation is dependent on the connexin isoform providing gap junctional coupling.
2007,
Pubmed
Zatechka,
Studies of the mitogen-activated protein kinases and phosphatidylinositol-3 kinase in the lens. 1. The mitogenic and stress responses.
2002,
Pubmed
Zhang,
Gap junction-mediated intercellular biochemical coupling in cochlear supporting cells is required for normal cochlear functions.
2005,
Pubmed
