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Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides.

Kanaporis G, Mese G, Valiuniene L, White TW, Brink PR, Valiunas V - J. Gen. Physiol. (2008)

Bottom Line: However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA.

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 and 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 (I(h)) 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 I(h) 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 approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = 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.

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(A–C) Plots of junctional conductance versus time from Cx43 (•, n = 9), Cx40 (○, n = 12), and Cx26 (▾, n = 12) expressing cell pairs, measured during the application of cAMP to the donor cell. Data represent means ± SEM. (D) Current recorded from the single SpIH-expressing cell with 20 μM cAMP in the pipette in perforated patch mode. There was no detectable SpIH current increase in perforated patch mode, and SpIH current increased dramatically when perforated patch was converted to the conventional whole cell patch clamp (around 15 min time mark). See text for details. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine.
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fig5: (A–C) Plots of junctional conductance versus time from Cx43 (•, n = 9), Cx40 (○, n = 12), and Cx26 (▾, n = 12) expressing cell pairs, measured during the application of cAMP to the donor cell. Data represent means ± SEM. (D) Current recorded from the single SpIH-expressing cell with 20 μM cAMP in the pipette in perforated patch mode. There was no detectable SpIH current increase in perforated patch mode, and SpIH current increased dramatically when perforated patch was converted to the conventional whole cell patch clamp (around 15 min time mark). See text for details. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine.

Mentions: An important control experiment is the determination of cAMP effects, if any, on junctional conductance. Junctional conductance was monitored in all experiments using a Vj step of 10 mV with a duration of 50 ms. Fig. 5 shows summarized plots of gjversus time for cell pairs expressing Cx43, Cx26, or Cx40, measured during the delivery of cAMP to the donor cell. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine. Over the time course of the experiment, no statistically significant alteration in junctional conductance was observed for Cx43, Cx40, or Cx26. This is in apparent contradiction to previous studies that showed a rapid (minutes) increase in junctional conductance of ∼25% for Cx40 transfected into SKHep1 cells using dual whole cell patch (Van Rijen et al., 2000). We used a perforated patch mode with one cell of the pair (recipient cell), which might represent a reason for the apparent difference. It is also quite possible that HeLa cells do not possess the same molecular machinery as SKHep1 cells, since we were not able to distinguish any difference on junctional conductance between IBMX- and 2’5′dideoxyadenosine-treated or nontreated cell pairs.


Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides.

Kanaporis G, Mese G, Valiuniene L, White TW, Brink PR, Valiunas V - J. Gen. Physiol. (2008)

(A–C) Plots of junctional conductance versus time from Cx43 (•, n = 9), Cx40 (○, n = 12), and Cx26 (▾, n = 12) expressing cell pairs, measured during the application of cAMP to the donor cell. Data represent means ± SEM. (D) Current recorded from the single SpIH-expressing cell with 20 μM cAMP in the pipette in perforated patch mode. There was no detectable SpIH current increase in perforated patch mode, and SpIH current increased dramatically when perforated patch was converted to the conventional whole cell patch clamp (around 15 min time mark). See text for details. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279171&req=5

fig5: (A–C) Plots of junctional conductance versus time from Cx43 (•, n = 9), Cx40 (○, n = 12), and Cx26 (▾, n = 12) expressing cell pairs, measured during the application of cAMP to the donor cell. Data represent means ± SEM. (D) Current recorded from the single SpIH-expressing cell with 20 μM cAMP in the pipette in perforated patch mode. There was no detectable SpIH current increase in perforated patch mode, and SpIH current increased dramatically when perforated patch was converted to the conventional whole cell patch clamp (around 15 min time mark). See text for details. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine.
Mentions: An important control experiment is the determination of cAMP effects, if any, on junctional conductance. Junctional conductance was monitored in all experiments using a Vj step of 10 mV with a duration of 50 ms. Fig. 5 shows summarized plots of gjversus time for cell pairs expressing Cx43, Cx26, or Cx40, measured during the delivery of cAMP to the donor cell. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine. Over the time course of the experiment, no statistically significant alteration in junctional conductance was observed for Cx43, Cx40, or Cx26. This is in apparent contradiction to previous studies that showed a rapid (minutes) increase in junctional conductance of ∼25% for Cx40 transfected into SKHep1 cells using dual whole cell patch (Van Rijen et al., 2000). We used a perforated patch mode with one cell of the pair (recipient cell), which might represent a reason for the apparent difference. It is also quite possible that HeLa cells do not possess the same molecular machinery as SKHep1 cells, since we were not able to distinguish any difference on junctional conductance between IBMX- and 2’5′dideoxyadenosine-treated or nontreated cell pairs.

Bottom Line: However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA.

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 and 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 (I(h)) 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 I(h) 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 approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = 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.

Show MeSH
Related in: MedlinePlus