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

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 αß 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 α-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.

Reconstitution of surface lipoprotein translocation through the Slam translocon.

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machinery required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a SLP TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1-containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

Cryo-EM structure of an open conformation of a gap junction hemichannel in lipid bilayer nanodiscs.

To mediate cell-to-cell communication via gap junction channels (GJCs), connexins (Cx) traffic as hexameric hemichannels to the plasma membrane, which dock end-to-end between adjacent cell membranes, thereby forming a dodecameric intercellular conduit. Hemichannels also function independently to mediate the passage of contents between the cytoplasm and extracellular space. To generate hemichannels, the mutation N176Y was introduced into the second extracellular loop of Cx26. The electron cryomicroscopy structure of the hexameric hemichannel in lipid bilayer nanodiscs displays an open pore and a 4-helix bundle transmembrane design that is nearly identical to dodecameric GJCs. In contrast to the high resolution of the transmembrane α-helices, the extracellular loops are less well resolved. The conformational flexibility of the extracellular loops may be essential to facilitate surveillance of hemichannels in apposed cells to identify compatible Cx isoforms that enable intercellular docking. Our results also provide a structural foundation for previous electrophysiologic and permeation studies of Cx hemichannels.

A Steric "Ball-and-Chain" Mechanism for pH-Mediated Regulation of Gap Junction Channels.

Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric "ball-and-chain" gating particle.

Structure/Function Analysis of human ZnT8 (SLC30A8): A Diabetes Risk Factor and Zinc Transporter.

The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic ß-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn2+/H+ antiporter. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/ß structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.