Figures and data
Cx26K125E gap junctions are constitutively closed at a PCO2 of 35 mmHg.
a,b) Montages each showing bright field DIC image of HeLa cells with mCherry fluorescence superimposed (leftmost image) and the permeation of NBDG from the recorded cell to coupled cells. Yellow arrow indicates the presence of a gap junction between the cells; scale bar, 20 µm. The numbers are the time in minutes following the establishment of the whole cell recording. In Cx26WT expressing cells (a), dye rapidly permeates into the coupled cell. For Cx26K125E expressing cells (b), no dye permeates into the neighbouring cell even after 10 minutes of recording despite the presence of a morphological gap junction. c) Quantification of fluorescence intensity in the recorded cell (donor) and the potentially coupled cell (recipient) for both Cx26WT and Cx26K125E (7 pairs of cells recorded for WT and K125E, data presented as mean ± SEM). While dye permeation to the recipient cell follows the entry of dye into the donor for Cx26WT, no dye permeates to the acceptor cell for Cx26K125E. Note that the fluorescent intensity in the donor cell for Cx26K125E is higher than for Cx26WT, presumably because the dye remains trapped in the donor cell rather than diffusing to the acceptor cell.
Comparison of WT Cx26 with the K125E mutant.
a) Superposition of density for WT90 Cx26 (PDB ID 7QEQ; pink) on the density for K125E90 (blue). The ovals show the position of TM2 from each subunit and the arrows show the direction of the difference between TM2 in the wild type and mutant structures. b) As (a) but focussed on TM2. c) Density associated with one subunit of the K125E90 structure (unsharpened map). The structure has been coloured according to residue number with blue at the N-terminus and red at the C-terminus. The K125VRIEG130 motif has been depicted in magenta. d) Superposition of the WT90 structure (7QEQ, pink) on the K125E90-1 structure (based on colouring in (c)). The positions of E125 and R104 have been depicted by magenta and cyan spheres respectively with the hypothesised salt bridge between them marked with a line. e) As d but focussed on TM1. The position of TM1 differs between the two structures as seen by the change in position of Trp24. (HC denotes the hydrocarbon chain from a lipid.)
Distinct classes from classification of Cx26 solubilised in LMNG.
a) Overall density associated with LMNG90-class2. b) As (a) focussed on the KVRIEG motif and the link between the N-terminus and TM1. Arg98 and Arg99 of TM2 appear to stabilise the conformation of the KVREIG motif of the same subunit, with Arg98 interacting with Glu129 and Arg99 stabilising the main chain. c) Density associated with LMNG90-class1. d) Superposition of the density from (a) and (c).
Comparison of structures refined from most distinct classes from LMNG classification
a) Overall superposition showing the movement of TM2 and the link between the N-terminus and TM1. LMNG90-class2 in cyan and LMNG90-class1 in yellow (alternate subunits have been coloured in lighter shades). The KVRIEG motif has been coloured magenta with a sphere indicating the position of K125. The residues between the N-terminus and TM1 for the LMNG90-class2 structure have been coloured pink. b) As (a) but focussed on TM1.
Focussed classification of a single subunit results in density for the cytoplasmic loop consistent with models from Alphafold.
a) Models generated by Alphafold for a single subunit (left; coloured according to confidence level) and for the hexamer (right; in wheat with the position of K125 depicted by blue sticks and the position of R104 in red). b) Focussed classification of a single subunit (coloured as in Figure 2 and highlighted by an oval) resulted in clear density for part of the cytoplasmic loop in a conformation consistent with the models from Alphafold. This does not extend to the neighbouring subunits. c) Superposition of the single subunit built into the density (cyan) on the alphafold model (wheat). Showing the change in position of the helix in the cytoplasmic loop (highlighted by an arrow in the relevant colour). d) Reconstituting a hexamer by replicating the conformation of the subunit seen in (b) to all 6 subunits of the hexamer results in an apparently more closed conformation of the hemichannel, though there are also residue clashes, especially at the N-terminus. Lys 125 and Arg 104 are depicted with red and blue sticks respectively.
Comparison of the two structures derived from the LMNG classification with other structures of connexins.
a) Superposition of a single subunit from the LMNG90-class2 (cyan) and LMNG90-class1 (yellow) structures on: Cx26 crystal structure (chartreuse, PDB ID 2ZW3); Cx50 (white, 7JJP); Cx43 in gate closing (red, 7XQF), flexible intermediate (chocolate, 7XQI) and pore lining (salmon, 7F94) conformations; Cx36 in pore lining (pink, 7XNH) and flexible N terminus (raspberry, 7XKT). The structures were superposed based on all chains of the hexamer. Only TM1, TM2 and the N-terminal helices are shown for each structure. b) View from the cytoplasmic side of the hemichannels from the same structures as in (a). Only the cytoplasmic part of the four transmembrane helices are shown. c) As (a) for the Cx26 (left), Cx43 (middle) and Cx36 (right) structures separately. Trp 24 in each of the Cx26 and Cx43 structures has been depicted with a sphere representation. The sequence identities for common residues to Cx26 is 49% for Cx50, 43% for Cx43 and 35% for Cx36.
Schematic representation of conformational changes
Schematic view of the cytoplasmic region of two opposing subunits within one hemichannel of the gap junction. The open structure on the left and the constricted structure on the right are in a dynamic equilibrium. The introduction of a negative charge on Lys125 of the KVRIEG motif (magenta) pushes the equilibrium to the right. In going from one conformation to the other: ① the cytoplasmic region of TM2 flexes around Phe83 and the cytoplasmic loop adopts a more defined conformation; ② the cytoplasmic region of TM1 rotates, illustrated here by the movement of Trp24; ③ the N-terminal helix, which will be affected by both these conformational changes adopts a position within the pore that constricts entrance to the channel.
Cryo-EM data collection and processing statistics
Cryo-EM refinement and validation statistics
Workflow for processing of cryo-EM data for K125E sample in CO2/HCO3- buffer.
The star denotes the classifications with the appearance of the PCN conformation that refine to a resolution greater than 4Å.
Density for the transmembrane and N-terminal helix associated with each structure.
Workflows for processing of cryo-EM data for samples in HEPES buffer
Comparison of density maps from wild type and K125E Cx26 purified in HEPES buffer at pH 7.4:
a) WT Cx26 at 4.9Å resolution sharpened with a B-factor of -100. b) K125E sharpened with B-factor of -273 and low pass filtered to 5Å. c) Superposition of the two maps.
Workflow for processing of cryo-EM data for K125R sample in CO2/HCO3- buffer
The star denotes the classifications with the appearance of the PCN conformation that refine to a resolution greater than 4Å.
Non-protein density in pore of Cx26.
a) Density for K125E90. b) LMNG90 D6 averaged map superposed on the map for K125E90.
Workflow for initial processing of cryo-EM data for WT sample, solubilised in LMNG in CO2/HCO3- buffer.
The star denotes the classifications with the appearance of the PCN conformation that refine to a resolution greater than 4Å.
