Biology subjects

Lovatt, A.

Publications and source records attributed to Lovatt, A..

3 recordsLinked to original sources

bioRxiv2025

CO2 sensitive connexin channel synapses in the VTA release 5HT to regulate dopaminergic neurons

Hypercapnic arousal is a life-preserving reflex that promotes awakening in response to elevated PCO2, yet the cellular mechanisms by which CO2 is detected remain incompletely understood. Serotonergic neurons of the dorsal raphe (DR) have been implicated in hypercapnic responses, but the molecular sensor that directly detects CO2 within this circuit is unknown. Here, we show that the CO2-gated connexin 26 (Cx26) hemichannel is expressed on serotonergic terminals projecting from the DR to the ventral tegmental area (VTA). Brief elevations in PCO2 modulated the excitability of VTA dopamine neurons through serotonergic signaling without altering glutamatergic transmission, and these effects were abolished by pharmacological inhibition of hemichannels. By expressing Cx26 and genetically encoded GRAB5HT sensors in cultured cells, we further demonstrate that Cx26 hemichannels opened by elevated PCO2 are directly permeable to serotonin. Selective removal of CO2 sensitivity from Cx26 in DR serotonergic neurons delayed arousal from sleep during transient hypercapnia and reduced activation of VTA dopamine neurons in vivo. Together, these findings identify Cx26 hemichannels as a direct CO2 sensor that enables serotonin release through a channel co-synapse and contributes to hypercapnic arousal.

neuroscience↗

bioRxiv2025

On the mechanisms of permselectivity of connexin hemichannels to small molecules

Connexins can either that act as hemichannels, to facilitate ion and small molecule movement from the cytosol to the extracellular space or as gap junction channels to provide a pathway for solute exchange between cells. Connexins are ubiquitously expressed throughout the body and are implicated in a wide range of processes. The permselectivity of connexin hemichannels for small neurochemicals remains poorly understood. By coexpressing genetically encoded fluorescent sensors for ATP, glutamate and lactate with a range of connexins, we examined the ability of different hemichannels to permit release of these compounds under physiological conditions and in response to physiological stimuli (small changes in PCO2 and transmembrane depolarisation). We found that some connexin hemichannels were relatively non-selective (Cx26, Cx32, Cx43, Cx31.1) allowing passage of ATP, glutamate and lactate. By contrast other connexin hemichannels (Cx36, Cx46 and Cx50) were highly selective. Cx36 and Cx46 hemichannels allowed release of ATP, but not glutamate or lactate. The size of the permeating molecule cannot be the sole determinant of permselectivity. By contrast, Cx50 hemichannels permitted the release of lactate and glutamate but not ATP. We also found that the nature of the opening stimulus could alter the permselectivity of the hemichannel-for some of the relatively non-selective connexins, hemichannel opening via depolarisation was ineffective at allowing release of lactate. By performing a mutational analysis, informed by the differential selectivity of the closely related Cx46 and Cx50 hemichannels, we found that the charge on the N-terminus and N-terminus-TM2 interactions are key contributors to permselectivity for ATP.

cell biology↗

bioRxiv2025

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.

cell biology↗