Electron Microscopic Analysis of Lens Gap Junction Connexin50 Hemichannels and Channels

Abstract

The mammalian lens is an avascular organelle that depends on gap junction (GJ) mediated intercellular communication for homeostasis, growth and development. Connexin50 (Cx50) is one of three lens GJ proteins and one of the largest members of the connexin protein family. Cx50 is present in all cells of the lens, first synthesized in the epithelium and persisting at high levels in differentiating fibers. Cases of human hereditary cataracts caused by >20 Cx50 distinct mutations demonstrate its crucial role in lens function and Cx50 knockout mice develop cataracts. Little is known about Cx50 structure except through homology with Cx26, a small connexin solved to 3.5 Å resolution by X‐ray crystallography and NMR of Cx50 C‐terminal peptides. Cx26 and Cx50 have strong sequence conservation and folding in their transmembrane and extracellular domains, but Cx26 lacks the extensive, non‐conserved C‐terminus present in Cx50. We have examined the full‐length Cx50 hemichannels and channels by single particle EM methods, the only structural biology technique that directly addresses how the large Cx50 C‐terminus is organized within the full‐length channel. Using baculovirus expression in Sf9 cells, we have purified both full‐length mouse Cx50 (440 amino acids) and a truncated form (Cx50del250‐440, 249 amino acids long) missing most of the C‐terminus such that it is slightly longer than Cx26 (226 amino acids). Electron micrographs of purified Cx50 hemichannels show a range of morphologies due to the way the oligomers are oriented on the grid. In contrast, Cx26 and Cx50del250‐440 channels are more homogeneous, displaying the canonical “doughnut‐like” appearance in projection because their structure.Funding: NIH grants GM065937 (GS) & GM103412 (NCMIR)