CO₂-dependent opening of Connexin 43 hemichannels

Valentin-Mihai Dospinescu
Alexander Mascarenhas
Jack Butler
Sarbjit Nijjar
Kyara de Oliveira Taborda
Sean Connors
Lumei Huang
Nicholas Dale author has email address

School of Life Sciences, University of Warwick, Coventry, United Kingdom

https://doi.org/10.7554/eLife.105989.1

Open access
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Figures and data

Structures of Cx43 and Cx26 predicted by AF3.
(A) α- and β-connexin sequence alignment with the known CO₂-sensitive connexins at the top, Cx26, Cx30 and Cx32, followed by the α-connexin Cx43. The carbamylation motif is highlighted by the green box. (B) Cx26 and Cx43 AlphaFold3 structural alignments. (C) Cx26 and Cx43 hemichannel structural prediction.

Cx43 hemichannels can be opened by an increase in PCO₂.
The CO₂ sensitivity of Cx43 was assayed using three different methods. (A) Dye-loading with CBF from 20 (left) to 70 mmHg PCO₂ (right). The inset represents the 0 Ca²⁺ control, white scale bar is 20 μm. Cumulative probability graph represents all the data points measured with more than 200 cells per condition, box plot shows the medians from each independent transfection. (B) Electrophysiology voltage patch clamp was done on HeLa cells expressing Cx43. The baseline was set to 35 mmHg PCO₂ and three different concentrations were used (20, 55, 70 mmHg). The conductance change evoked by a change in PCO₂ was measured and plotted as a dose response curve (right side, median with interquartile range) fitted by the Hill equation H=6, EC₅₀=43 mmHg. (C) The genetically-encoded ATP sensor GRAB_ATP was co-transfected alongside Cx43 into HeLa cells (n=22). Representative images at different PCO₂ concentrations of the cells are shown, grey scale bar is 20 μm. Traces from the recording the images were selected from are shown; each line represent a different cell, the fluorescence was normalised by dividing all values by the baseline median pixel intensity before plotting. Box plot shows the µM ATP release as determined by normalisation to the 3 µM control solution applied. (D) HeLa cells transfected with GRAB_ATP alone do not exhibit CO₂ dependent ATP release (n=14), summary box plot shows the µM ATP release as determined by normalisation to 3 µM ATP calibration.

Cx43 gap junction channels are insensitive to changes in PCO₂.
A) DIC image with mCherry fluorescence superimposed. Gap junctions are evident as red stripes between cells (yellow arrows). The fluorescence images show the patch pipette filled with NBDG its loading into the recorded cell and the neighbouring cells coupled via gap junctions. The numbers in each bottom left corner are the time of the recording from whole cell breakthrough in minutes. Scale bar 20 µm. B) Summary graphs showing the time for the acceptor cell to reach 10% of the fluorescence of the donor cell at three different levels of PCO₂ (n=6 for each condition).

The effect of single Lys mutations on the CO₂ sensitivity of Cx43.
HeLa cells transfected with the mutant variant of Cx43 were subjected to the dye-loading protocol. Images of dye loading in response to CO₂ and the 0 Ca²⁺ positive control, together with the cumulative probability plots are shown for K105Q (A), K109Q (B), K144Q (C), K234Q (D). (E) AlphaFold3 prediction of Cx43 with hypothesised carbamylation bridge residues highlighted – K234 in orange and K109 in red. (F) Summary box plots for dye-loading showing the change in median pixel intensity from 20 mmHg PCO₂ for each condition (70 mmHg PCO₂ – green and 0 Ca²⁺ blue, n=5) for WT (recalculated data from Fig 2A for comparison) and each mutation. Scale bars are 20 µm.

The effect of single Lys mutations on CO₂-dependent ATP release from Cx43.
The genetically-encoded ATP sensor GRAB_ATP was co-transfected alongside Cx43 mutant variations, K105Q (A) n=23, K109Q (B) n=20, K144Q (C) n=12, K234Q (D) n=15. Representative trace measurements were selected; each line represents a different cell; the fluorescence was normalised by dividing all values by the baseline median pixel intensity before plotting. Box plot on the right (E) and (F) represent the µM ATP released as determined through normalisation to the 3 µM ATP application.

The effect of single Lys mutations on the CO₂ dose-response properties of Cx43.
Comparison of Cx43^WT (data replotted from Fig 2C), Cx43^K105Q, Cx43^K109Q (data replotted from Fig 4E) and Cx43^K144Q (data replotted from Fig 4F). Data is plotted as medians with lower and upper quartiles. For the WT, K105Q and K144Q the fitted curve is drawn according to a modified Hill equation:
Where K and H are the affinity and Hill coefficient of the channel for opening by CO₂, K_i and H_i are the affinity and Hill coefficient for inhibition of the channel by CO₂, and Max_ATP is the asymptotically maximum release of ATP to CO₂. For K109Q which did not exhibit CO₂-dependent inhibition at high levels of PCO₂, the Hill equation was used. The parameters for the curves are:

The effect of single Lys mutations on the CO₂ dose-response properties of Cx43.
Comparison of Cx43^WT (data replotted from Fig 2C), Cx43^K105Q, Cx43^K109Q (data replotted from Fig 4E) and Cx43^K144Q (data replotted from Fig 4F). Data is plotted as medians with lower and upper quartiles. For the WT, K105Q and K144Q the fitted curve is drawn according to a modified Hill equation:
Where K and H are the affinity and Hill coefficient of the channel for opening by CO₂, K_i and H_i are the affinity and Hill coefficient for inhibition of the channel by CO₂, and Max_ATP is the asymptotically maximum release of ATP to CO₂. For K109Q which did not exhibit CO₂-dependent inhibition at high levels of PCO₂, the Hill equation was used. The parameters for the curves are:

Paired Lys mutations are required to abolish the CO₂ sensitivity of Cx43.
Expressing cells pixel intensity for each construct was measured from 5 individual transfections, with at least 40 cells per condition. Representative images for all combinations tried are shown – K109Q K144Q (A), K105Q K144Q (B), K105Q K109Q (C), K105Q K234Q (D), K144Q K234Q (E). Cumulative probability plots display all measured data points. The box plot shows the change in median pixel intensity from 20 mmHg PCO₂ for each transfection for 70 mmHg PCO₂ (green) and 0 Ca²⁺ (blue).

Paired Lys mutations abolish CO₂ dependent ATP release via Cx43.
HeLa cells co-expressing Cx43 and GRAB_ATP were subjected to changing PCO₂-levels and fluorescence was recorded . Representative traces (A-E) for each of the Cx43 mutations – K105Q K144Q (A) n=23, K109Q K144Q (B) n=19, K105Q K234Q (C) n=17, K109Q K234Q (D) n=16, K144Q K234Q (E) n=13. The traces indicate normalised fluorescence changes (ΔF/F₀). 3 µM ATP was applied at the end of each experiment to confirm sensor functionality. Furthermore, as a positive control, 50 mM KCl was applied to depolarise the cells and confirm channel function. (F-G) Box plots summarising the total ATP release in µM for each double mutant in 55 mmHg PCO₂ and 50 mM KCl. Data points represent individual measured cells.

Introduction of negative charge into the carbamylation motif via Lys to Glu mutations has mixed effects on CO₂ sensitivity of Cx43 hemichannels.
Expressing cells pixel intensity for each construct was measured from 5 individual transfections, with at least 40 cells per condition. (A-C) Representative cell images for 20 (left), 70 (right) with insets displaying the 0 Ca²⁺ control for each mutant K105E (A), K144E (B), K234E (C). Scale bar represents 20 µm. Cumulative probability graphs of pixel intensities are shown on the right for each mutant with three conditions 20 mmHg PCO₂ (blue line), 70 mmHg PCO₂ (orange) and 0 Ca²⁺ (grey line). The box plot shows change in median pixel intensity from 20 mmHg PCO₂ for each transfection for 70 mmHg PCO₂ (green boxes) and 0 Ca²⁺ (blue boxes).

Lys to Glu mutations abolish CO₂-dependent ATP Release.
GRAB_ATP fluorescence traces of ATP release from cells expressing single mutant connexins: K105E (A) n=18, K144E (B) n=17, K234E (C) n=13, in response to 55 mmHg PCO₂ (red bars), 50 mM KCl depolarisation control (orange bars) and a 3 µM ATP control application at the end of all experiments to confirm sensor functionality. Traces show changes in normalised fluorescence over time (ΔF/F), indicating ATP release. On the right, the box plots display the quantified ATP release in µM for each mutant under 55 mmHg PCO₂ and 50 mM KCl. Each data point represents a measurement from an individual cell.

The Cx43 double mutant, K105E K109E, is constitutively open.
(A) Representative fluorescence images of cells expressing the Cx43 K105E K109E mutant under low (20 mmHg PCO₂, left) and high CO₂ (70 mmHg PCO₂, right), an inset showing the 0 Ca²⁺ control is present. Scale bar = 20 µm. Cumulative probability plot of pixel intensity for each condition is shown on the right, overall indicating higher baseline fluorescence and perpetually open hemichannels. (B) Dye-loading with 20 mmHg PCO₂ (left) and 20mmHg PCO₂ with 100 µm LaCl₃. Cumulative probability plot for pixel intensity under these conditions is shown on the right. (C) Fluorescence traces of ATP release from cells co-expressing GRAB_ATP and Cx43 K105E K109E (n=16) under 20 mmHg PCO₂ and 20 mmHg PCO₂ with 100 µM LaCl₃ (red bar). Fluorescence is normalised to baseline. (D) Fluorescence traces of ATP release from cells co-expressing GRAB_ATP and Cx43 WT under 20 mmHg PCO₂ and 20 mmHg PCO₂ with 100 µM LaCl₃ (red bar). (E) Box plots summarizing median pixel intensity under the different conditions, showing a significant reduction in intensity in the presence of LaCl₃ (p = 0.03). (F) Box plot shows normalised fluorescence changes values for the difference between – 20 mmHg PCO₂ (representing baseline normalisation) and the application of LaCl₃ for both the K105E K109E and WT Cx43 constructs.

PCO₂-dependent modulation of amplitude of fEPSPs in hippocampus is mediated via Cx43.
(A-C) Time-course plots showing the average amplitude of the normalised fEPSP (± SEM) amplitude in response to different conditions. Inserts display representative fEPSPs. (A) Control condition showing an increase in fEPSP amplitude in response to a modest change to PCO₂ (20 to 35 mmHg), red bar represent the application of 35 mmHg PCO₂, baseline conditions – 20 mmHg PCO₂. (B) EPSP responses in the presence of Gap26 (blue bar) and subsequent wash. (C) EPSP responses with scrambled Gap26 peptide applied (blue bar). (D) Box plots representing the normalised fEPSP size difference (baseline was subtracted) with colour coded conditions: orange – control 35 mmHg PCO₂, green – Gap26 with pre (35G), after (35S Wash).

Pathological mutations of Cx43 remove its sensitivity to CO₂.
A,B) L90V prevents CO₂-dependent dye loading. The dye loading assay shows no change in fluorescence between 20 and 70 mmHg, yet functional channels are expressed as shown by the zero Ca²⁺ positive control. Box plot shows the change in median pixel intensity from 20 mmHg PCO₂ for each transfection for 70 mmHg PCO₂ (green boxes) and 0 Ca²⁺ (blue boxes). C-E) GRAB_ATP recordings shown that L90V and A44V also abolish CO₂-dependent ATP release.

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