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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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Plots of the SpIH activation time versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40 (○) in the absence (A) and presence (B) of phosphodiesterase (IBMX) and adenylate cyclase inhibitors (2’,5′ dideoxyadenosine). The SpIH activation times were reduced in IBMX and 2’5′dideoxyadenosine-treated cells and were reciprocally proportional to junctional conductance. The solid lines correspond to the first order regression and the dashed lines are the 95% confidence intervals. The arrows pointing to 15 and 20 nS indicate the threshold for detection of cAMP in recipient cell for Cx26 and Cx40, respectively.
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fig4: Plots of the SpIH activation time versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40 (○) in the absence (A) and presence (B) of phosphodiesterase (IBMX) and adenylate cyclase inhibitors (2’,5′ dideoxyadenosine). The SpIH activation times were reduced in IBMX and 2’5′dideoxyadenosine-treated cells and were reciprocally proportional to junctional conductance. The solid lines correspond to the first order regression and the dashed lines are the 95% confidence intervals. The arrows pointing to 15 and 20 nS indicate the threshold for detection of cAMP in recipient cell for Cx26 and Cx40, respectively.

Mentions: We also compared cells under physiological conditions without IBMX and 2’5′dideoxyadenosine with those in a pool of IBMX and 2’5′dideoxyadenosine-exposed preparations to assess whether or not there was significant degradation of cAMP over the time course of our experiments. For this analysis we measured time t (see Fig. 2 B and Fig. 3 B), for the SpIH current to reach saturation in the recipient cell (i.e., ∼20 μM of cAMP) after cAMP delivery to the donor cell. Fig. 4 shows the datasets for non-IBMX–treated and IBMX-treated cell pairs plotted versus gj for all three connexins. These data show that there is a reduction in the delivered cAMP without inhibition of phospodiesterase. The time to SpIH current saturation is reduced in IBMX and 2’5′dideoxyadenosine-treated cells for all three connexins, indicating that the effective free pool of cAMP delivered to the reporter gene SpIH is reduced if IBMX and 2’5′dideoxyadenosine are not present. Interestingly, we found that without IBMX or 2’5′dideoxyadenosine, cAMP transfer was detected only in well-coupled cell pairs of Cx26 (>15 nS) and Cx40 (>20 nS). In Fig. 4 A the arrows pointing to 15 and 20 nS on the X axis indicate the minimal coupling to detect cAMP transfer for Cx26 and Cx40, respectively.


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)

Plots of the SpIH activation time versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40 (○) in the absence (A) and presence (B) of phosphodiesterase (IBMX) and adenylate cyclase inhibitors (2’,5′ dideoxyadenosine). The SpIH activation times were reduced in IBMX and 2’5′dideoxyadenosine-treated cells and were reciprocally proportional to junctional conductance. The solid lines correspond to the first order regression and the dashed lines are the 95% confidence intervals. The arrows pointing to 15 and 20 nS indicate the threshold for detection of cAMP in recipient cell for Cx26 and Cx40, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279171&req=5

fig4: Plots of the SpIH activation time versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40 (○) in the absence (A) and presence (B) of phosphodiesterase (IBMX) and adenylate cyclase inhibitors (2’,5′ dideoxyadenosine). The SpIH activation times were reduced in IBMX and 2’5′dideoxyadenosine-treated cells and were reciprocally proportional to junctional conductance. The solid lines correspond to the first order regression and the dashed lines are the 95% confidence intervals. The arrows pointing to 15 and 20 nS indicate the threshold for detection of cAMP in recipient cell for Cx26 and Cx40, respectively.
Mentions: We also compared cells under physiological conditions without IBMX and 2’5′dideoxyadenosine with those in a pool of IBMX and 2’5′dideoxyadenosine-exposed preparations to assess whether or not there was significant degradation of cAMP over the time course of our experiments. For this analysis we measured time t (see Fig. 2 B and Fig. 3 B), for the SpIH current to reach saturation in the recipient cell (i.e., ∼20 μM of cAMP) after cAMP delivery to the donor cell. Fig. 4 shows the datasets for non-IBMX–treated and IBMX-treated cell pairs plotted versus gj for all three connexins. These data show that there is a reduction in the delivered cAMP without inhibition of phospodiesterase. The time to SpIH current saturation is reduced in IBMX and 2’5′dideoxyadenosine-treated cells for all three connexins, indicating that the effective free pool of cAMP delivered to the reporter gene SpIH is reduced if IBMX and 2’5′dideoxyadenosine are not present. Interestingly, we found that without IBMX or 2’5′dideoxyadenosine, cAMP transfer was detected only in well-coupled cell pairs of Cx26 (>15 nS) and Cx40 (>20 nS). In Fig. 4 A the arrows pointing to 15 and 20 nS on the X axis indicate the minimal coupling to detect cAMP transfer for Cx26 and Cx40, respectively.

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