Three-Dimensional Structure of a Recombinant Gap Junction Membrane Channel

Abstract

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Gap junction channel structure in the early 21st century: facts and fantasies

“…Twodimensional projection maps at 7 Å resolution revealed superimposed α-helices that could only arise if the connexons are rotationally staggered by 30° around the 6-fold symmetry axis [5] ( Figure 1a). Thereafter, a 3D map at 7.5 Å in-plane resolution showed that each connexon contains 24 rod-like densities readily interpreted as transmembrane α-helices [7] (Figure 1). The primary sequence identity of each transmembrane helix could not be assigned at this resolution, so they were arbitrarily designated A, B, C and D (Figure 2).…”

“…While there must be some structural differences to account for different limiting pore diameters and charge selectivities, it would be truly remarkable if the fundamental organization and packing of the transmembrane helices were different. More to the point, the transmembrane densities derived from cryo-EM of the M34A mutant of Cx26 [9] are virtually identical to those derived from cryo-EM of Cx43 [7].…”

“…The packing of the 24 α-helices within the 6-fold symmetric connexon can accommodate several possible molecular boundaries. Scrutiny of the density map and exclusion of models that require crossovers of the E1 and E2 loops suggests that the most likely molecular boundaries are a closely-packed 4-helix bundle or a more loosely packed "checkmark" arrangement [7], shown in Figure 2 for the helical assignments of Fleishman et al [8] (blue) and Skerrett et al [43] (green). A map at high resolution will be required to resolve these possibilities.…”

“…Support for this model is suggested by recent cryoEM studies of the M34A mutant of Cx26 [9]. In contrast to previous cryoEM studies of twodimensional crystals derived from native plasma membranes [5,7,8], these two-dimensional crystals were generated by reconstituting detergent-solubilized, purified, recombinant Cx26 into lipid bilayers. Surprisingly, the connexons appeared to redock during the reconstitution, thereby forming dodecameric channels (Figure 4).…”

“…The primary tools for structure analysis of gap junction channels include electron microscopy and image analysis [4][5][6][7][8][9], X-ray diffraction [10][11][12], nuclear magnetic resonance (NMR) spectroscopy [13][14][15] and atomic force microscopy (AFM) [16][17][18]. Mutagenic, biochemical and electrophysiological approaches have also been used to elucidate the structure-function relationships of gap junction channels.…”