CO₂-dependent opening of Connexin 43 hemichannels

Reviewer #1 (Public review):

Summary:

This study builds on previous work demonstrating that several beta connexins (Cx26, Cx30, and Cx32) have a carbamylation motif which renders them sensitive to CO2. In response to CO2, hemichannels composed of these connexins open, enabling diffusion of small molecules (such as ATP) between the cytosol and extracellular environment. Here, the authors have identified that an alpha connexin, Cx43, also contains a carbamylation motif, and they demonstrate that CO2 opens Cx43 hemichannels. Most of the study involves using transfected cells expressing wild-type and mutant Cx43 to define amino acids required for CO2 sensitivity. Hippocampal tissue slices in culture were used to show that CO2-induced synaptic transmission was affected by Cx43 hemichannels, providing a physiological context. The authors point out that the Cx43 gene significantly diverges from the beta connexins that are CO2 sensitive, suggesting that the conserved carbamylation motif was present before the alpha and beta connexin genes diverged.

Strengths:

(1) The molecular analysis defining the amino acids that contribute to the CO2 sensitivity of Cx43 is a major strength of the study. The rigor of analysis was strengthened by using three independent assays for hemichannel opening: dye uptake, patch clamp channel measurements, and ATP secretion. The resulting analysis identified key lysines in Cx43 that were required for CO2-mediated hemichannel opening. A double K to E Cx43 mutant produced a construct that produced hemichannels that were constitutively open, which further strengthened the analysis.

(2) Using hippocampal tissue sections to demonstrate that CO2 can influence field excitatory postsynaptic potentials (fEPSPs) provides a native context for CO2 regulation of Cx43 hemichannels. Cx43 mutations associated with Oculodentodigital Dysplasia (ODDD) inhibited CO2-induced hemichannel opening, although the mechanism by which this occurs was not elucidated.

Weaknesses:

(1) Cx43 channels are sensitive to cytosolic pH, which will be affected by CO2. Cytosolic pH was not measured, and how this affects CO2-induced Cx43 hemichannel activity was not addressed.

(2) Cultured cells are typically grown in incubators containing 5% CO2, which is ~40 mmHg. It is unclear how cells would be viable if Cx43 hemichannels are open at this PCO2.

(3) Experiments using Gap26 to inhibit Cx43 hemichannels in fEPSP measurements used a scrambled peptide as a control. Analysis should also include Gap peptides specifically targeting Cx26, Cx30, and Cx32 as additional controls.

(4) The mechanism by which ODDD mutations impair CO2-mediated hemichannel opening was not addressed. Also, the potential roles for inhibiting Cx43 hemichannels in the pathology of ODDD are unclear.

(5) CO2 has no effect on Cx43-mediated gap junctional communication as opposed to Cx26 gap junctions, which are inhibited by CO2. The molecular basis for this difference was not determined.

(6) Whether there are other non-beta connexins that have a putative carbamylation motif was not addressed. Additional discussion/analysis of how the evolutionary trajectory for Cx43 maintaining a carbamylation motif is unique for non-beta connexins would strengthen the study.

Reviewer #2 (Public review):

Summary:

This paper examines the CO2 sensitivity of Cx43 hemichannels and gap junctional channels in transiently transfected Hela cells using several different assays, including ethidium dye uptake, ATP release, whole cell patch clamp recordings, and an imaging assay of gap junctional dye transfer. The results show that raising pCO2 from 20 to 70 mmHg (at a constant pH of 7.3) causes an increase in opening of Cx43 hemichannels but does not block Cx43 gap junctions. This study also showed that raising pCO2 from 20 to 35 mm Hg resulted in an increase in synaptic strength in hippocampal rat brain slices, presumably due to downstream ATP release, suggesting that the CO2 sensitivity of Cx43 may be physiologically relevant. As a further test of the physiological relevance of the CO2 sensitivity of Cx43, it was shown that two pathological mutations of Cx43 that are associated with ODDD caused loss of Cx43 CO2-sensitivity. Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26. To understand the structural changes involved in CO2 sensitivity, a number of mutations were made in Cx43 sites thought to be the equivalent of those known to be involved in the CO2 sensitivity of Cx26, and the CO2 sensitivity of these mutants was investigated.

Strengths:

This study shows that the apparent lack of functional Cx43 hemichannels observed in a number of previous in vitro function studies may be due to the use of HEPES to buffer the external pH. When Cx43 hemichannels were studied in external solutions in which CO2/bicarbonate was used to buffer pH instead of HEPES, Cx43 hemichannels showed significantly higher levels of dye uptake, ATP release, and ionic conductance. These findings may have major physiological implications since Cx43 hemichannels are found in many organs throughout the body, including the brain, heart, and immune system.

Weaknesses:

(1) Interpretation of the site-directed mutation studies is complicated. Although Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26, the results of site-directed mutation studies were inconsistent with a simple model in which K144 and K105 interact following carbamylation to cause the opening of Cx43 hemichannels.

(2) Secondly, although it is shown that two Cx43 ODDD-associated mutations show a loss of CO2 sensitivity, there is no evidence that the absence of CO2 sensitivity is involved in the pathology of ODDD.

Reviewer #3 (Public review):

In this paper, the authors aimed to investigate carbamylation effects on the function of Cx43-based hemichannels. Such effects have previously been characterized for other connexins, e.g., for Cx26, which display increased hemichannel (HC) opening and closure of gap junction channels upon exposure to increased CO2 partial pressure (accompanied by increased bicarbonate to keep pH constant).
The authors used HeLa cells transiently transfected with Cx43 to investigate CO2-dependent carbamylation effects on Cx43 HC function. In contrast to Cx43-based gap junction channels that are reported here to be insensitive to PCO2 alterations, they provide evidence that Cx43 HC opening is highly dependent on the PCO2 pressure in the bath solution, over a range of 20 up to 70 mmHg encompassing the physiologically normal resting level of around 40 mmHg. They furthermore identified several Cx43 residues involved in Cx43 HC sensitivity to PCO2: K105, K109, K144 & K234; mutation of 2 or more of these AAs is necessary to abolish CO2 sensitivity. The subject is interesting and the results indicate that a fraction of HCs is open at a physiological 40 mmHg PCO2, which differs from the situation under HEPES buffered solutions where HCs are mostly closed under resting conditions. The mechanism of HC opening with CO2 gassing is linked to carbamylation, and the authors pinpointed several Lys residues involved in this process.

Overall, the work is interesting as it shows that Cx43 HCs have a significant open probability under resting conditions of physiological levels of CO2 gassing, probably applicable to the brain, heart, and other Cx43 expressing organs. The paper gives a detailed account of various experiments performed (dye uptake, electrophysiology, ATP release to assess HC function) and results concluded from those. They further consider many candidate carbamylation sites by mutating them to negatively charged Glu residues. The paper ends with hippocampal slice work showing evidence for connexin-dependent increases of the EPSP amplitude that could be inhibited by HC inhibition with Gap26 (Figure 10). Another line of evidence comes from the Cx43-linked ODDD genetic disease, whereby L90V as well as the A44V mutations of Cx43 prevented the CO2-induced hemichannel opening response (Figure 11). Although the paper is interesting, in its present state, it suffers from (i) a problematic Figure 3, precluding interpretation of the data shown, and (ii) the poor use of hemichannel inhibitors that are necessary to strengthen the evidence in the crucial experiment of Figure 2 and others.