scholarly journals Mechanism of v-Src- and mitogen-activated protein kinase-induced reduction of gap junction communication

2003 ◽  
Vol 284 (2) ◽  
pp. C511-C520 ◽  
Author(s):  
G. Trevor Cottrell ◽  
Rui Lin ◽  
Bonnie J. Warn-Cramer ◽  
Alan F. Lau ◽  
Janis M. Burt
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Electrical Coupling ◽  
Mitogen Activated Protein Kinase ◽  
Dye Coupling ◽  
Open Probability ◽  
Unitary Conductance ◽  
Gap Junction Communication ◽  
Mitogen Activated Protein ◽  
Cx 43

Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.

scholarly journals Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures.

1987 ◽  
Vol 105 (1) ◽  
pp. 541-551 ◽  
Author(s):  
D C Spray ◽  
M Fujita ◽  
J C Saez ◽  
H Choi ◽  
T Watanabe ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Electrical Coupling ◽  
Sulfated Polysaccharides ◽  
Sulfate Proteoglycan ◽  
Dye Coupling ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
In Cells

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.

scholarly journals Properties of Single Voltage-gated Proton Channels in Human Eosinophils Estimated by Noise Analysis and by Direct Measurement

2003 ◽  
Vol 121 (6) ◽  
pp. 615-628 ◽  
Author(s):  
Vladimir V. Cherny ◽  
Ricardo Murphy ◽  
Valerij Sokolov ◽  
Richard A. Levis ◽  
Thomas E. DeCoursey
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Power Spectral Analysis ◽  
Open Probability ◽  
Proton Channels ◽  
Voltage Gated ◽  
Unitary Conductance ◽  
Power Spectral ◽  
Current Events ◽  
Channel Currents

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi ≤ 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{{\sigma}}_{\mathrm{H}}^{\mathrm{2}}\) \end{document}. We analyze \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{{\sigma}}_{\mathrm{H}}^{\mathrm{2}}\) \end{document} data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi ≤ 5.5 near Vthreshold (the voltage at which gH turns on), single channel–like current events were observed with amplitudes 7–16 fA.

Biophysical characterization of zebrafish connexin35 hemichannels

2004 ◽  
Vol 287 (6) ◽  
pp. C1596-C1604 ◽  
Author(s):  
Virginijus Valiunas ◽  
Rickie Mui ◽  
Elizabeth McLachlan ◽  
Gunnar Valdimarsson ◽  
Peter R. Brink ◽  
...  
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Biophysical Characterization ◽  
Whole Cell ◽  
Gap Junction Channels ◽  
N2a Cells ◽  
Unitary Conductance ◽  
Cell Depolarization

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 Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 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 Cx35 hemichannels ( Ihc) that were activated and increased by stepwise depolarization of membrane potential ( Vm) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance ( γhc: 250–320 pS). The γhc of Cx35 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 Cx35 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.

scholarly journals Gap Junctions Regulate Extracellular Signal-regulated Kinase Signaling to Affect Gene Transcription

2005 ◽  
Vol 16 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Joseph P. Stains ◽  
Roberto Civitelli
Keyword(s):  
Protein Kinase ◽  
Gap Junctions ◽  
Gap Junction ◽  
Gene Transcription ◽  
Electrical Coupling ◽  
Mitogen Activated Protein Kinase ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Extracellular Signal ◽  
Gap Junctional

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Iα1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.

Persistence of gap junction communication during myocardial ischemia

2001 ◽  
Vol 280 (6) ◽  
pp. H2563-H2571 ◽  
Author(s):  
Marisol Ruiz-Meana ◽  
David Garcia-Dorado ◽  
Sinead Lane ◽  
Pilar Pina ◽  
Javier Inserte ◽  
...  
Keyword(s):  
Myocardial Ischemia ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Electrical Coupling ◽  
Atp Depletion ◽  
Mediated Communication ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Gap Junction Communication ◽  
Chemical Coupling

During myocardial ischemia, severe ATP depletion induces rigor contracture followed by intracellular Ca2+ concentration ([Ca2+]i) rise and progressive impairment of gap junction (GJ)-mediated electrical coupling. Our objective was to investigate whether chemical coupling through GJ allows propagation of rigor in cardiomyocytes and whether it persists after rigor development. In end-to-end connected adult rat cardiomyocytes submitted to simulated ischemia the interval between rigor onset was 3.7 ± 0.7 s, and subsequent [Ca2+]i rise was virtually identical in both cells, whereas in nonconnected cell pairs the interval was 71 ± 12 s and the rate of [Ca2+]i rise was highly variable. The GJ blocker 18α-glycyrrhetinic acid increased the interval between rigor onset and the differences in [Ca2+]i between connected cells. Transfer of Lucifer yellow demonstrated GJ permeability 10 min after rigor onset in connected cell pairs, and 30 min after rigor onset in isolated rat hearts submitted to nonflow ischemia but was abolished after 2 h of ischemia. GJ-mediated communication allows propagation of rigor in ischemic myocytes and persists after rigor development despite acidosis and increased [Ca2+]i.

scholarly journals Characterization of the Mitogen-activated Protein Kinase Phosphorylation Sites on the Connexin-43 Gap Junction Protein

1996 ◽  
Vol 271 (7) ◽  
pp. 3779-3786 ◽  
Author(s):  
Bonnie J. Warn-Cramer ◽  
Paul D. Lampe ◽  
Wendy E. Kurata ◽  
Martha Y. Kanemitsu ◽  
Lenora W. M. Loo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
Connexin 43 ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Junction Protein ◽  
Mitogen Activated Protein ◽  
Gap Junction Protein ◽  
Kinase Phosphorylation

scholarly journals Conductance and Permeability of the Residual State of Connexin43 Gap Junction Channels

2002 ◽  
Vol 119 (2) ◽  
pp. 171-186 ◽  
Author(s):  
Feliksas F. Bukauskas ◽  
Angele Bukauskiene ◽  
Vytas K. Verselis
Keyword(s):  
Gap Junction ◽  
Fluorescent Protein ◽  
Single Channel ◽  
Electrical Coupling ◽  
Dye Transfer ◽  
Channel Pore ◽  
Gap Junction Channels ◽  
Gating Mechanism ◽  
Gated Channel ◽  
Green Fluorescent

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell–cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (Vj) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher Vjs that are negative on the side of gated hemichannel. The degree of rectification increases when Cl− is replaced by Asp− and decreases when K+ is replaced by TEA+. These data are consistent with an increased anionic selectivity of the residual state. The Vj-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast Vj-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.

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