Connexin50 hemichannels are opened by CO2: implications for lens physiology

Connexin50 (Cx50) is expressed in lens fibre cells. As mutations of Cx50 cause cataracts its physiological role in the lens must be important. We have used recent cryoEM structures of Cx50 and the predictive power of Alphafold3 to identify the presence of a carbamylation motif, originally described in Cx26, that suggests that Cx50 might be CO2 sensitive. By expressing the naturally truncated version of Cx50 in HeLa cells and utilising coexpression of genetically encoded sensors iGluSnFr or eLACCO1.1, we have demonstrated the CO2-dependent opening of Cx50 hemichannels, and their permeability to lactate and glutamate. By mutating the two key residues of the carbamylation motif, K105 and K140, we have shown that the motif is required for CO2 sensitivity. Mutations of the residue V44 cause cataracts and these mutations abolish the CO2 sensitivity of Cx50. Using Fluo-4 Ca2+ imaging with lens slices we have demonstrated CO2-dependent Ca2+ influxes into fibre cells that are blocked by La3+ and exhibit the same CO2 dose dependence as Cx50 hemichannels. Lens fibre cells respond to glutamate via NMDA receptors and our data shows that the Ca2+ influx to raised PCO2 partially depends on NMDA receptor activation. We hypothesize that CO2-dependent gating of Cx50, subsequent release of glutamate resulting in the downstream activation of glutamate receptors, and the consequent alterations in transmembrane Na+ fluxes, provide homeostatic control of the microcirculation system that is critical for lens health.