Kanaporis G et al. (2008), Gap junction channels exhibit connexin-specific...

ECB-ART-40662

J Gen Physiol 2008 Apr 01;1314:293-305. doi: 10.1085/jgp.200709934.

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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 .

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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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Species referenced: Echinodermata
Genes referenced: hcn3 LOC100887844 LOC100893907 LOC115919910 LOC586260

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	Figure 1. Properties of SpIH channels. (A) Voltage protocol (Vm) and whole cell currents (Im) recorded in control, untransfected (top), and SpIH-transfected HeLa cells in the absence (middle) or presence (bottom) of 50 μM cAMP. Im were elicited by 2-s voltage pulses delivered from a holding potential of 0 mV to test potentials between −20 and −120 mV, in 20-mV increments, and returning to a tail potential of +50 mV. Im increased in SpIH-transfected cells when cAMP was present in the pipette solution. (B) Average of tail current densities measured after voltage step to Vm = −100 mV from control cells (0.3 ± 0.05 pA/pF, n = 7), SpIH-transfected cells (5.3 ± 0.7 pA/pF, n = 13), and SpIH-transfected cells with 50 μM cAMP in the pipette (28.6 ± 3.1 pA/pF, n = 13). Data represent means ± SEM. (C) Dependence of the SpIH current on intracellular cAMP concentration. Solid line represents best fit of data to the Hill equation with EC50 = 7.1 μM, Hill slope = 2.01. Data represent means ± SEM, obtained from a total 48 cells at Vm = −100 mV. The experiments were performed in the presence of IBMX and 2’,5′ dideoxyadenosine.
	Figure 2. 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 (*).
	Figure 3. (A and B) Plots of normalized tail current versus time recorded from the recipient cells after cAMP injection into the source cell for cell pairs expressing Cx43 (•): gj = 19 nS (A), and 2 nS (B); Cx26 (▾): gj = 25 nS (A), and 8 nS (B); and Cx40(○): gj = 22 nS (A) and 10 nS (B). SpIH activation time, t corresponds to the time when SpIH current reaches saturation in the recipient cell. The first order regression over the linear part of the plots (dashed line shown in Cx40 plot, A) was used to determine current activation rate ΔI/Δt. (C) Summarized plots of the SpIH tail current activation rate (ΔI/Δt) versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40(○). The solid lines correspond to the first order regressions with the following slopes: (30.0 ± 2.5) · 10−4 s−1/nS for Cx43, (7.3 ± 0.6) · 10−4 s−1/nS for Cx26, and (4.8 ± 1.1) · 10−4 s−1/nS for Cx40. The dashed lines are the 95% confidence intervals. The experiments were performed in the presence of IBMX and 2’,5′ dideoxyadenosine. Insert: current activation rate (ΔI/Δt) versus channel number for all three connexins with slopes of (1.6 ± 0.1) · 10−4 s−1/channel for Cx43 (•), (0.8 ± 0.07) · 10−4 s−1/channel for Cx26 (▾), and (0.6 ± 0.1) · 10−4 s−1/channel for Cx40 (○).
	Figure 4. 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.
	Figure 5. (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.
	Figure 6. Summary of LY flux data versus junctional conductance for Cx43, Cx26, and Cx40 cell pairs. Each data point for Cx26 (▾) represents a recipient cell fluorescence intensity over injected cell fluorescence intensity 12 min after LY injection (Valiunas et al., 2002). The solid lines correspond to the first order regressions and the dashed lines represent confidence intervals (95%) for each plot. See text for references on derivation of regression lines for Cx40 and Cx43.
	Figure 7. Single channel properties of Cx26 channels. Voltage protocol (top) and single channel currents recorded from homotypic Cx26 cell pairs with pipette solutions of different ionic compositions. Top: K+ aspartate− pipette solution. Transjuctional voltage Vj = 110 mV, the current histograms yielded γj of 111pS/109 pS for negative/positive Vj. Bottom: Na+ aspartate− pipette solution, Vj = 90 mV, γj = 83 pS/85 pS for negative/positive Vj.
	Figure 8. Selectivity properties of Cx43, Cx40, and Cx26 to various solutes relative to K+. The bars represent Na+/K+, cAMP/K+, and LY/ K+ ratios plotted on log scale for Cx43 (white bar), Cx26 (gray bar), and Cx40 (black bar).
	Figure 9. Levitt model plotting D(x)/Do versus pore diameter for cAMP (○) and LY (•), where D(x) is the diffusion coefficient for a solute within the channel and Do is the equivalent within the cytoplasm. D(x)/Do is assumed to be approximated by the calculated flux ratios for cAMP/K+ and LY/K+. The continuous and dashed lines represent cAMP/K and LY/K+ ratios for Cx43 and Cx40, respectively.

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