PH-Gating of GAP Junction Channels: Visualization of a "Ball-and-chain" by Cryo-EM

Gap junction channels (GJCs) mediate intercellular communication between adjacent cells and are formed by the end-to-end docking of two hexameric hemichannels comprised of connexin (Cx) subunits. The central, axial pore enables the exchange of hydrated ions, second messengers and metabolites up to ∼15 Å in diameter. A variety of physiological processes and pathological states affect channel activity, including intracellular Ca2+, pH, membrane potential, and trans-junctional voltage (Vj). Here we used single-particle electron cryo-microscopy (cryoEM) to examine the conformational changes associated with pH-mediated regulation of GJCs. Recombinant human Cx26 was purified as dodecameric GJCs, which remained homogenous and monodisperse when examined at pH 7.5 and 6.4. In general, the structures of Cx26 GJCs reconstituted in amphipol recapitulated the authentic Cx43 GJC in native membranes at 5.7 Å resolution and our previous Cx26 X-ray crystal structure at 3.8 Å resolution. Within the transmembrane and extracellular domains, the map resolution was ∼3.5 Å, whereas the cytoplasmic loop and cytoplasmic tail were less well-resolved, likely due to conformational flexibility. 3D classification of images recorded from particles at pH 6.4 yielded two conformations, one resembling the open physiological pH structure and another with a pore-occluding density. Modeling of the polypeptide into the cryoEM maps suggested that acidic pH elicits a helix-coil transition and association of the amino-termini to form the gating particle. To our knowledge, this is the first instance in which a complete, oligomeric “ball-and-chain” has been visualized.