Connexin-hemichannels-mediated ATP release causes lung injury following chlorine inhalation

Chlorine (Cl₂) is a highly reactive halogen gas that undergoes rapid hydrolysis in lung epithelial lining fluid (ELF) upon inhalation, forming hypochlorous acid (HOCl) and hydrochloric acid (HCl). These products subsequently, through chemical reactions, modify the structure and the function of membrane proteins. Herein, we investigated the effects of Cl₂ on connexin-hemichannels and the release of ATP in the ELF. Adult C57BL/6 mice were subjected to 400 ppm Cl₂ for 30 min. Subsequent analysis revealed a marked increase in ATP levels within the BAL, with concentrations reaching 43.952 ± 9.553 nM at 2 h and 30.554 ± 7.383 nM at 24 h post exposure, relative to control. In addition, at 24 h post exposure, the lung wet/dry (W/D) ratio significantly increased from 4.48 ± 0.142 to 5.067 ± 0.359, whereas alveolar fluid clearance (AFC) decreased from 0.249 ± 0.019 to 0.145 ± 0.018. Electrophysiological recordings in alveolar type 2 (AT2) cells revealed reduced open probabilities (P_o) of both ENaC (4 pS) and a cation channel (18 pS), declining from 0.323 ± 0.021 and 0.202 ± 0.022 to 0.151 ± 0.042 and 0.091 ± 0.019, respectively. Instillation of 50 µL of 100 µg/mL Gap27-a connexin mimetic peptide selectively inhibiting connexin-hemichannels-administered 30 min post exposure, restored ATP to control, normalized the W/D ratio, improved AFC, and reestablished ENaC function. Moreover, Gap27 normalized airway resistance following methacholine challenge. In human airway smooth muscle cells (hASMCs), 100 μM ATP induced [Formula: see text] elevation and depolarized <i>V</i>_m to -40 mV, with both effects partially reversed by P2X₇R inhibitor, A804598.<b>NEW & NOTEWORTHY</b> Inhaled chlorine gas reacts with lung epithelial lining fluid to form hypochlorous and hydrochloric acids that alter membrane protein structure and function. Under oxidative stress, connexin hemichannels open, releasing ions and metabolites, such as ATP. The released ATP signals danger, cell death, and tissue injury. Early administration of Gap27, a connexin-hemichannel inhibitor, at 30 min post exposure preserves ENaC function and prevents the subsequent development of pulmonary edema. These compelling findings underscore a promising therapeutic strategy.