Biology subjects

Nijjar, S.

Publications and source records attributed to Nijjar, S..

4 recordsLinked to original sources

bioRxiv2026

Deep evolutionary origins of the connexin gene family

The vertebrate connexin family encodes a diverse repertoire of transmembrane channels that mediate both intercellular communication and cytosol to extracellular space exchange. Their apparent absence in non-chordates has led to the prevailing view that connexins arose during chordate evolution with analogous functions performed in non-chordates by the innexins, a structurally similar but non-homologous protein family. Here, using a combination of sequence and structural analyses, we discover the presence of connexins in anthozoan and protostome genomes despite widespread secondary gene loss in Bilateria. We show that these connexins can be present in up to 100 copies per genome and are often found as N-terminal domains of NOD-like receptors (NLRs) or other immune genes. We show that these invertebrate connexins localise at the cell membrane, function as either hemichannels (anthozoans and protostomes) or gap junctions (protostomes), and respond to depolarisation enabling the transfer of small molecules. Further, we demonstrate that a subset of the invertebrate connexin-like domains are sensitive to CO2 and mirroring findings in vertebrate connexins, the CO2-mediated gating stems from the formation of a carbamate bridge between neighbouring subunits. Taken together, we trace the evolution of this gene family to the cnidarian-bilaterian ancestor, demonstrate functional similarity between invertebrate and vertebrate connexins, and suggest an ancient role of connexins in innate immunity.

evolutionary biology↗

bioRxiv2025

CO2-dependent opening of Connexin 43 hemichannels

Sequence and structure comparisons between alpha and beta connexins, Cx43 and Cx26, revealed that Cx43 has a motif, the carbamylation motif, that confers CO2-sensitivity on a subset of beta connexins. By using a fluorescent dye loading assay, whole cell patch clamp recordings and real time measurement of ATP release via GRABATP we have demonstrated that Cx43 hemichannels open in a highly CO2 sensitive manner over the range 20 to 70 mmHg. Mutational analysis confirms that the equivalent residues to those in Cx26 that have been shown to be involved in mediating the effects of CO2 on gating of hemichannels and gap junction channels, also mediate Cx43 hemichannel gating. Our data predicts that Cx43 will be partially open and able to release ATP at resting physiological levels of PCO2. We have tested this prediction in acute hippocampal slices, by showing that CO2-dependent enhancement of synaptic transmission can be blocked by the Cx43-selective mimetic peptide Gap26. Our data resolves an inconsistency in the literature between in vivo studies suggesting that Cx43 hemichannels are at least partially open at rest, and in vitro studies, performed in the absence of HCO3-/CO2 buffering that show Cx43 hemichannels are shut. Our evidence suggests that the ancestral gene that duplicated to give the alpha and beta connexin clades must have possessed the carbamylation motif. CO2 sensitivity is thus a fundamental ancient characteristic of several connexins that has been lost in more recently derived members of this gene family.

neuroscience↗

bioRxiv2023

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

biochemistry↗

bioRxiv2019

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

Cx26 hemichannels open in response to moderate elevations of CO2 (PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2. Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 5.6 nS, 95% confidence interval, 3.2 to 11.9 nS) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification, but using 30 mM propionate we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 5.3 nS (2.8 to 8.3 nS). This effect of propionate was unaffected by the K125R mutation (median reduction 7.7 nS, 4.1 to 11.0 nS). pH-dependent and CO2-dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the Keratitis Ichthyosis Deafness syndrome (N14K, A40V and A88V) also abolished the CO2-dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound, is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

cell biology↗