Abstract
Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.
Highlights
Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma
Ca2 þ overload closes GJCs, which likely contributes to the electrical instability and arrhythmias that occur during myocardial ischaemia
Molecular replacement (MR) with a dimer of a Cx Ca model based on our cryoEM map (PDB ID 1TXH5) resulted in X-ray crystallographic maps of sufficient quality to determine the helix directions and refine the relative orientations of the monomers
Summary
Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. Hemichannels from adjacent cells dock to form intercellular, dodecameric gap junction channels (GJCs; Fig. 1b), which can cluster to form gap junction plaques in the plasma membrane (Fig. 1c)[1,2] These dense, quasi-hexagonal arrays enabled electron microscopy of isolated plaques and low-resolution, three-dimensional, image reconstruction[3]. GJCs are responsible for homo- and hetero-cellular communication in vascular endothelial and smooth muscle cells, enabling orchestrated cellular activity over long distances in response to rapidly changing tissue demands[10] Owing to their diverse and specialized functions, Cx isoforms have highly variable selectivities, including moderate to significant charge preferences and distinct permeabilities to signalling molecules such as ATP and inositol triphosphate (IP3), with a molecular weight (MW) cutoff of about 1,000 Da and a size limit of up to B14 Å (refs 11,12). Ca2 þ overload closes GJCs, which likely contributes to the electrical instability and arrhythmias that occur during myocardial ischaemia
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