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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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Detection of intercellular transfer of cAMP. (A) Cell pair where one cell expressed SpIH-eGFP (green cell) and the other expressed RFP only (red cell). 500 μM cAMP was delivered via patch pipette into the red cell that did not express SpIH and SpIH currents were recorded from cell expressing SpIH in perforated patch mode. Both cells express Cx43. (B) Current recorded from the SpIH-expressing cell, in response to voltage pulses from a holding potential of 0 to −100 mV, returning to a tail potential of +50 mV. The arrow indicates the opening of the patch into whole cell recording mode and the beginning of cAMP delivery into the donor cell. SpIH current over time increased to steady state. The red bar on time axis indicates SpIH current activation time interval t during which current reaches saturation.(C) Segments of SpIH current records when the whole cell patch was opened (*) and 135 s later with saturated current (**).
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fig2: Detection of intercellular transfer of cAMP. (A) Cell pair where one cell expressed SpIH-eGFP (green cell) and the other expressed RFP only (red cell). 500 μM cAMP was delivered via patch pipette into the red cell that did not express SpIH and SpIH currents were recorded from cell expressing SpIH in perforated patch mode. Both cells express Cx43. (B) Current recorded from the SpIH-expressing cell, in response to voltage pulses from a holding potential of 0 to −100 mV, returning to a tail potential of +50 mV. The arrow indicates the opening of the patch into whole cell recording mode and the beginning of cAMP delivery into the donor cell. SpIH current over time increased to steady state. The red bar on time axis indicates SpIH current activation time interval t during which current reaches saturation.(C) Segments of SpIH current records when the whole cell patch was opened (*) and 135 s later with saturated current (**).

Mentions: The detection of cAMP transfer between cells of a pair was accomplished by coculturing HeLa or N2A cell pairs where one cell expressed eGFP and SpIH, while the other expressed RFP only (green and red cells, respectively in Fig. 2 A). This allowed assessment of cAMP transfer by monitoring the activity of SpIH current while measuring junctional conductance using dual patch clamp. In this configuration one pipette was in whole cell mode and contained 500 μM cAMP and the recipient cell, containing SpIH, was in perforated patch mode. An example of the activation of SpIH current in recipient cell is shown in Fig. 2 B. Currents recorded from the SpIH-expressing cell were derived in response to voltage pulses from a holding potential of 0 to −100 mV and returning to a tail potential of +50 mV. SpIH currents increased over time to a new steady-state value due to cAMP diffusion from cell 2 to cell 1. Examples of the SpIH current activation for the record shown in Fig. 2 B are shown in Fig. 2 C, one at the time the whole cell patch was opened (*) and the other 135 s later when the SpiH current became saturated with cAMP from cell 2 (**).


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)

Detection of intercellular transfer of cAMP. (A) Cell pair where one cell expressed SpIH-eGFP (green cell) and the other expressed RFP only (red cell). 500 μM cAMP was delivered via patch pipette into the red cell that did not express SpIH and SpIH currents were recorded from cell expressing SpIH in perforated patch mode. Both cells express Cx43. (B) Current recorded from the SpIH-expressing cell, in response to voltage pulses from a holding potential of 0 to −100 mV, returning to a tail potential of +50 mV. The arrow indicates the opening of the patch into whole cell recording mode and the beginning of cAMP delivery into the donor cell. SpIH current over time increased to steady state. The red bar on time axis indicates SpIH current activation time interval t during which current reaches saturation.(C) Segments of SpIH current records when the whole cell patch was opened (*) and 135 s later with saturated current (**).
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Related In: Results  -  Collection

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fig2: Detection of intercellular transfer of cAMP. (A) Cell pair where one cell expressed SpIH-eGFP (green cell) and the other expressed RFP only (red cell). 500 μM cAMP was delivered via patch pipette into the red cell that did not express SpIH and SpIH currents were recorded from cell expressing SpIH in perforated patch mode. Both cells express Cx43. (B) Current recorded from the SpIH-expressing cell, in response to voltage pulses from a holding potential of 0 to −100 mV, returning to a tail potential of +50 mV. The arrow indicates the opening of the patch into whole cell recording mode and the beginning of cAMP delivery into the donor cell. SpIH current over time increased to steady state. The red bar on time axis indicates SpIH current activation time interval t during which current reaches saturation.(C) Segments of SpIH current records when the whole cell patch was opened (*) and 135 s later with saturated current (**).
Mentions: The detection of cAMP transfer between cells of a pair was accomplished by coculturing HeLa or N2A cell pairs where one cell expressed eGFP and SpIH, while the other expressed RFP only (green and red cells, respectively in Fig. 2 A). This allowed assessment of cAMP transfer by monitoring the activity of SpIH current while measuring junctional conductance using dual patch clamp. In this configuration one pipette was in whole cell mode and contained 500 μM cAMP and the recipient cell, containing SpIH, was in perforated patch mode. An example of the activation of SpIH current in recipient cell is shown in Fig. 2 B. Currents recorded from the SpIH-expressing cell were derived in response to voltage pulses from a holding potential of 0 to −100 mV and returning to a tail potential of +50 mV. SpIH currents increased over time to a new steady-state value due to cAMP diffusion from cell 2 to cell 1. Examples of the SpIH current activation for the record shown in Fig. 2 B are shown in Fig. 2 C, one at the time the whole cell patch was opened (*) and the other 135 s later when the SpiH current became saturated with cAMP from cell 2 (**).

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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