Valiunas V et al. (2019), Lens Connexin Channels Have Differential Permea...

ECB-ART-47443

Invest Ophthalmol Vis Sci 2019 Sep 03;6012:3821-3829. doi: 10.1167/iovs.19-27302.

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Lens Connexin Channels Have Differential Permeability to the Second Messenger cAMP.

Valiunas V , Brink PR , White TW .

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Purpose: Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation. Methods: We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor 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 were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule. Results: cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46. Conclusions: These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.

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Species referenced: Echinodermata
Genes referenced: hcn3 LOC100887844 LOC100893907 LOC115919910 ptch1

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	Figure 1. An electrophysiologic assay for intercellular transfer of cAMP through gap junction channels. (A) When single SpIH expressing cells were whole-cell patched without cAMP in the pipette (cell on left), SpIH currents were small (black line). When cAMP was present in the patch pipette (cell on right), SpIH currents were increased 4- to 5-fold (red line). (B) Cell pairs were co-cultured where one cell expressed the connexin to be tested and SpIH (recipient cell on left, cell 1). The other cell expressed only the connexin to be tested (source cell on right, cell 2). Five hundred micromolar cAMP was delivered via a whole-cell patch pipette into cell 2, and SpIH currents were recorded from cell 1 in the perforated patch mode, before (black line), and after (red line) opening the patch on cell 2.
	Figure 2. Cx46, but not Cx50, mediated intercellular transfer of cAMP. (A) Data from a Cx46 cell pair with a gap junctional conductance of 31 nS are shown. The SpIH current recorded from cell 1 over time, in response to voltage pulses of 0 to 100 mV, returning to a tail potential of +50 mV. At time 0, the patch in cell 2 was opened initiating cAMP delivery. The SpIH tail current over time increased approximately 4-fold. (B) Cx50 failed to mediate transfer of cAMP. The SpIH current remained constant in the recipient cell after 500μM cAMP was released in the source cell. This Cx50 cell pair had a gap junctional conductance of 16 nS. (C) Expanded SpIH currents from when the whole cell patch was first opened (black asterisk/trace), and 280 seconds later (red asterisk/trace). The Cx46 cell pair showed an approximately 4-fold increase in SpIH tail currents. For the Cx50 cell pair, the SpIH tail currents were of the same magnitude, indicating little, or no, intercellular cAMP transfer through Cx50 channels. Data from a Cx43 cell pair with a conductance of 6 nS are shown for comparison.
	Figure 3. Quantitative comparison of intercellular cAMP transfer between the different lens connexins. (A) The change in the SpIH tail current was plotted versus time for individual cell pairs expressing Cx43 (filled circles), Cx46 (open triangles), or Cx50 (open circles). (B) A plot of the normalized tail current versus time recorded from the recipient cell after cAMP injection into the source cell for the Cx43 and Cx46 expressing cell pair. The SpIH activation time corresponds to the time when SpIH current reaches saturation in the recipient cell. The slopes of first order regressions over the linear part of the plot (dashed lines) were used to calculate the current activation rate (ΔI/Δt). (C) Expanded view of the SpIH tail current data for the Cx50 cell pair shown in (A). In the Cx50 cell pairs where the SpIH current increased, it never reached saturation during the finite period that cells could be recorded from, so ΔI/Δt was estimated from fitting the current increase over the entire range of data available.
	Figure 4. Summary quantification of SpIH current and gap junction conductance data. (A) The fold-increase in the SpIH tail current after cAMP delivery to the neighboring cell was plotted for individual cell pairs expressing Cx43 (filled circles, n = 9), Cx46 (open triangles, n = 7), or Cx50 (open circles, n = 10). Mean values of the data are plotted as horizontal lines, with standard deviations plotted as vertical error bars. (B) Plots of the time required for the SpIH tail current to reach saturation in Cx43 and Cx46 cell pairs. For Cx50, the time elapsed without the SpIH tail current reaching saturation was plotted. (C) Plots of the total gap junctional conductance measured between cell pairs expressing Cx43, Cx46, or Cx50.
	Figure 5. The SpIH current activation rate is dependent on the magnitude of gap junctional coupling between the cells. The SpIH tail current activation rate (ΔI/Δt) was plotted against gap junctional conductance for Cx43 (filled circles, n = 9), Cx46 (open triangles, n = 7), and Cx50 (open circles, n = 10). The solid lines correspond to first order regressions with the slopes given in the Table.
	Figure 6. Visualization of cyclic nucleotide passage through Cx43 and Cx50 gap junction channels using fluorescent ε-cAMP. (A) A pipette filled with 5 mM ε-cAMP was attached to a single cell within a group of HeLa cells expressing Cx43. After opening the patch in whole-cell mode, (B) dye transfer was detected to half of the neighboring cells within 5 minutes, and (C) a majority of the cells within 10 minutes. (D) In Cx50 transfected HeLa cells, (E, F) negligible transfer of ε-cAMP to neighboring cells was observed at 5 or 10 minutes following the opening of the patch pipette.
	Figure 7. Quantitative comparison of ε-cAMP flux through Cx43 and Cx50 channels. Simultaneous measurements of gap junction conductance and ε-cAMP flux were made in cell pairs. (A) In a Cx43 expressing cell pair with conductance of 12 nS, (B) high levels of ε-cAMP fluorescence were recorded in the recipient cell 5 minutes after the patch pipette was opened in the donor cell. (C) In a cell pair expressing Cx50 with a conductance of 16 nS, (D) very little passage of ε-cAMP into the recipient cell was seen after 5 minutes. (E) The mean (±SD) conductance for all tested Cx43 and Cx50 cell pairs (n = 3). (F) The ratio of ε-cAMP fluorescent intensity in the recipient cell compared with the donor cell for Cx43 and Cx50.

References [+] :

Abell, The role of adenosine 3',5'-cyclic monophosphate in the regulation of mammalian cell division. 1973, Pubmed