Abstract
Gap junction channels (GJC) form at cell-to-cell interfaces and are composed of hexameric hemichannels called connexons that dock end-to-end across the narrow extracellular gap. This allows direct intercellular exchange of ions and important metabolites up to ~15 Å in diameter. Connexins are the monomeric substituents of connexons and constitute a family of polytopic membrane proteins that have four transmembrane alpha-helices (TMD), two extracellular loops (E1 and E2), and three cytoplasmic domains (N-terminus (NT), intracellular loop (CL), and C-terminus (CT)).
GJCs are essential to most types of mammalian tissue, and channel dysfunction can lead to a wide variety of pathological defects. In the heart, cardiac gap junctions are responsible for the propagation of action potentials between adjacent myocytes by mediating intercellular ionic conduction. Consequently, they play a critical role in normal conduction and also in triggering fatal arrhythmias. In the cochlea, the Cx26 GJC plays a key role in K+ recycling, and over 100 point mutations to this channel result in sensorineural hearing loss. Cx26 GJCs and hemichannels also play an essential role in maintaining keratinocyte homeostasis, and dysfunction can result in several types of skin disease. In Schwann cells, the Cx32 GJC facilitates cytoplasmic continuity between myelin sheets, and mutations to any domain of this protein results in X-linked Charcot-Marie-Tooth disease (CMT).
In the scenario of myocardial infarction, anaerobic respiration leads to low intracellular pH, which inhibits GJCs. We used single-particle, electron cryo-microscopy (cryoEM) and mass spectrometry to identify conformational changes associated with pH-mediated regulation. The cryoEM structure of the Cx26 GJC at neutral pH recapitulated the previous GJC crystal structures. The GJC structure was well defined within the transmembrane and extracellular domains, whereas the N-terminal domains were conformationally heterogenous. Three-dimensional classification of particles at pH 6.4 revealed two conformational states, one resembling the physiological pH structure and another with pore-occluding densities. Lysine crosslinking with tandem mass spectrometry revealed closer association between the N-terminal domains and the cytoplasmic loops at acidic pH relative to neutral pH. The cryoEM maps suggests that acidic pH results in association and ordering of the N-terminal domains to form the “ball-and-chain” gating particle.
To investigate the hemichannel structure, we expressed and purified the Cx26 hemichannel by mutating a key connexon docking residue (N176Y) in the E2 loop. Using single-particle cryoEM, we reconstructed the hemichannel reconstituted in a nanodisc membrane mimetic to 4.3 Å resolution. Our cryoEM map recapitulates the TMD of previous GJC structures and appears to be in an open conformation suggesting that hemichannels are a constitutively open and require a stimulus to close in vivo.
In order to better understand Cx32 in the context of CMT, we expressed and purified Cx32 GJC with a C-terminal truncation at residue 250. Though the channel displayed aggregation upon concentration in a nanodisc or amphipol membrane mimetic, the protein displayed stable behavior in FA-3 detergent. The sample in detergent will now be vitrified in order to determine the structure by single-particle cryoEM.
To optimize protein purification conditions for future structural studies of the cardiac GJC Cx43, we have screened the expression and oligomeric state of several Cx43 orthologues using fluorescence-detection size-exclusion chromatography. While expression of Cx43 was successful in HEK293 cells, detergent solubilization resulted in the channel dissociating into connexin monomers. Using the structure of the Cx26 GJC, we engineered disulfide crosslinks in either the TMD or the extracellular domain to stabilize the oligomeric state of the channel. Crosslinks in the TMD resulted in enrichment of the hemichannel species, while crosslinks in the extracellular domain resulted in the enrichment of the full GJC. The extracellular domain crosslinks suggest that the Cx43 extracellular domains adopts a conformation similar to the Cx26 extracellular docking interface to form GJCs.
