Connexin Gap Junction Channels and Hemichannels: Insights from High-Resolution Structures

Simple Summary Gap junction channels are composed of an assembly of connexin proteins and allow direct communication between cells. They are highly conserved across vertebrates and form wide pores in cell membranes for the passage of ions and metabolites. Junctional channels are formed from the end-to-end docking of hemichannels, and both junctional channels and hemichannels are vital for many physiological activities. Several medical conditions are associated with problems in gap junction communication, ranging from deafness to fatal cardiac arrhythmias. Many connexin channel diseases can be linked to genetic mutations, and nearly 1000 have been identified in connexin genes. Prior to 2009, atomic-level structural details of gap junction channels were essentially non-existent. This information is critical for understanding channel function and to assess the pathological nature of disease-causing mutations. Fortunately, since 2009, the powerful tools of X-ray crystallography and electron cryomicroscopy have yielded over 50 high-resolution structures of connexin channels. This review aims to provide a comprehensive summary of this astounding 15-year period of structural discovery in the gap junction field. Divided into eight distinct sections, we describe key details found in this compendium of structures, such as conserved features in the design of connexin channels, insights into channel gating and surprises regarding where membrane lipids are bound to the channels. In addition, we highlight areas in which we need more information, such as the structure of highly flexible regions within connexin channels that have so far resisted visualization. Furthermore, targeting connexins for drug discovery is still in its infancy, and much more structural data are needed to pursue this end.Abstract Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an alpha beta fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a "ball-and-chain" fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein-protein and protein-lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane alpha-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation.