Connexin50 hemichannels are opened by CO2: implications for lens physiology.

Connexin50 (Cx50) is expressed in lens fiber cells. As mutations in Cx50 cause cataracts, its physiological role in the lens must be important. We have used recent cryo-EM 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 full-length and a naturally C-terminal truncated version of Cx50 in HeLa cells and utilizing coexpression of the genetically encoded sensors iGluSnFr or eLACCO1.1, we have demonstrated the CO2-dependent opening of both full-length and truncated Cx50 hemichannels. By mutating the two key residues of the carbamylation motif, K105 and K140, in the truncated version of Cx50, we have shown that the motif is required for the CO2 sensitivity of Cx50. Mutations of 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 fiber cells that are blocked by La3+ and exhibit the same CO2 dose dependence as Cx50 hemichannels. Lens fiber cells respond to glutamate via NMDA receptors, and our data show that the Ca2+ influx in response to raised PCO2 partially depends on NMDA receptor activation. We hypothesize that CO2-dependent gating of Cx50, the 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.