Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata.

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area - reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.

Microarray-based screening system identifies temperature-controlled activity of Connexin 26 that is distorted by mutations.

Here, we show that human Connexin 26 (hCx26 or Cx26WT) hemichannel opening rapidly enables the transport of small molecules when triggered by temperature and by compensation of the Ca2+ blockade with EDTA. Point mutations within Cx26 were analysed by a novel optical microarray-based Lucifer Yellow uptake assay or by two electrode voltage clamp (TEVC) on frog oocytes to monitor simultaneous activities of channel proteins. Point mutations L90P, F161S, R184P or K188N influenced the temperature-dependent activity drastically. Since several mutations blocked trafficking, the temperature-dependent activity of the recombinant synthesized and purified wild-type Cx26WT and Cx26K188N hemichannel was tested by liposome flux assay (LFA) and on a microarray-based Lucifer Yellow uptake assay under warm conditions (>30 °C). The data from TEVC measurements and dye flux experiments showed that the mutations gave no or only a weak activity at increased temperature (>30 °C). We conclude that the position K188 in the Cx26WT forms a temperature-sensitive salt bridge with E47 whereas the exchange to K188N destabilizes the network loop- gating filter, which was recently identified as a part of the flexible Ca2+ binding site. We assume that the temperature sensitivity of Cx26 is required to protect cells from uncontrolled release or uptake activities through Cx26 hemichannels.

Decreased phototoxicity of photodynamic therapy by Cx32/Cx26-composed GJIC: A "Good Samaritan" effect.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) has been widely used to treat malignant tumors. Our previous studies indicated that connexin (Cx) 32- and Cx26-composed gap junctional intercellular communication (GJIC) could improve the phototoxicity of PDT. However, the role of heterotypic Cx32/Cx26-formed GJIC in PDT phototoxicity is still unknown. Thus, the present study was aimed to investigate the effect of Cx32/Cx26-formed GJIC on PDT efficacy. METHODS: CCK8 assay was used to detect cell survival after PDT. Western blot assay was utilized to detect Cx32/Cx26 expression. "Parachute" dye-coupling assay was performed to measure the function of GJ channels. The intracellular Ca2+ concentrations were determined using flow cytometer. ELISA assay was performed to detect the intracellular levels of PGE2 and cAMP. RESULTS: The present study demonstrates there is a Cx32/Cx26-formed GJIC-dependent reduction of phototoxicity when cells were exposure to low concentration of Photofrin. Such a protective action is missing at low cell density due to the lack of GJ coupling. Under high-cell density condition, where there is opportunity for the cells to contact each other and form GJ, suppressing Cx32/Cx26-formed GJIC by either inhibiting the expression of Cx32/Cx26 or pretreating with GJ channel inhibitor augments PDT phototoxicity after cells were treated with at 2.5 µg/ml Photofrin. The above results suggest that at low Photofrin concentration, the presence of Cx32/Cx26-formed GJIC may decrease the phototoxicity of PDT, leading to the insensitivity of malignant cells to PDT treatment. The GJIC-mediated PDT insensitivity was associated with Ca2+ and prostaglandin E2 (PGE2 ) signaling pathways. CONCLUSION: The present study provides a cautionary note that for tumors expressing Cx32/Cx26, the presence of Cx32/Cx26-composed GJIC may cause the resistance of tumor cells to PDT. Oppositely, treatment strategies designed to downregulate the expression of Cx32/Cx26 or restrain the function of Cx32/Cx26-mediated GJIC may increase the sensitivity of malignant cell to PDT. Lasers Surg. Med. 51:301-308, 2019. © 2019 Wiley Periodicals, Inc.

Evolutionary adaptation of the sensitivity of connexin26 hemichannels to CO2.

CO2 readily combines with H2O to form [Formula: see text] and H+ Because an increase of only 100 nM in the concentration of H+ (a decrease of 0.1 unit of pH) in blood can prove fatal, the regulated excretion of CO2 during breathing is an essential life-preserving process. In rodents and humans, this vital process is mediated in part via the direct sensing of CO2 via connexin26 (Cx26). CO2 binds to hemichannels of Cx26 causing them to open and allow release of the neurotransmitter ATP. If Cx26 were to be a universal and important CO2 sensor across all homeothermic animals, then a simple hypothesis would posit that it should exhibit evolutionary adaptation in animals with different homeostatic set points for the regulation of partial pressure of arterial CO2 (PaCO2). In humans and rats, PaCO2 is regulated around a set point of 40 mmHg. By contrast, birds are able to maintain cerebral blood flow and breathing at much lower levels of PaCO2 Fossorial mammals, such as the mole rat, live exclusively underground in burrows that are both hypoxic and hypercapnic and can thrive under very hypercapnic conditions. We have therefore compared the CO2 sensitivity of Cx26 from human, chicken, rat and mole rat (Heterocephalus glaber). We find that both the affinity and cooperativity of CO2 binding to Cx26 have been subjected to evolutionary adaption in a manner consistent with the homeostatic requirements of these four species. This is analogous to the evolutionary adaptation of haemoglobin to the needs of O2 transport across the animal kingdom and supports the hypothesis that Cx26 is an important and universal CO2 sensor in homeotherms.