Plug Gating Mechanism of Gap Junction Channels Revealed by Electron Crystallography
Abstract: Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. It has been suggested by electrophysiological studies that gap junctions possess multiple gating mechanisms while only one structural model, a subunit rotation model, was proposed. Here we report the electron crystallographic structure of a connexin 26 mutant (Cx26M34A) and a projection structure of an N-terminus deletion of Cx26M34A missing amino acids 2-7 (Cx26M34Adel2-7). The three-dimensional map of Cx26M34A shows a prominent density in the pore of each hemichannel, suggesting that physical blockage may play an important role that underlies gap junction channel regulation. The projection map of Cx26M34Adel2-7 revealed that the plug density was dramatically reduced in comparison with that found in full length Cx26 channel. Our structures allow us to suggest that the two docked hemichannels can independently function and may regulate their activity autonomously with a plug that most likely consists of the connexin N-terminus in the vestibule. This gating model offers insights into an alternate mechanism on how gap junction channels gate in response to cellular stimuli.
Key words: gap junction, electron crystallography, cryo-electron microscopy, three dimensional structure analysis, two dimensional crystallization