Electron cryo-crystallography of a recombinant gap junction channel at 7a resolution: progress towards visualizing different conformational states

V. M. Unger*, D. W. Entri, Kn X. Guan, N. M. Kumar, N. B. Gilula, U. Feager

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Gap junction membrane channels mediate the electrical and metabolic coupling between cells. The channels are formed by the end-to-end docking of two hemi-channels (coimexons), each of which is formed by a hexameric cluster of protein subunits (connexins). The principal gap junction protein in the heart is a,connexin (Cx43). Detailed structural information is essential to delineate the molecular basis for channel gating. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated two-dimensional (2D) crystals of a recombinant, truncated ct,-connexin (0,0x2631). The projection map at ?A resolution revealed that each 30kD connexin subunit has a transmembrane a-helix that lines the aqueous pore and a second a-helix in close contact with the membrane lipids [Nature Struct. Biol 4:3943 (1997)]. Functional studies of BHK cells that express ce,Cx263T demonstrated that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of a,Cx263T channels. These results encouraged us to examine whether oleamide affects the structure of the channel. 2D crystals of a,Cx263T grown in the presence and absence of oleamide displayed diffraction to ~7A resolution. The crystals exhibit p6 symmetry with unit cell dimensions of a = b = 76.8A and y = 120". The 3D maps derived from tilted crystals display conservation in the hexameric arrangement of the connexon with four transmembrane cc-helices per subunit. Data collection and refinement are underway to characterize conformational changes associated with oleamide treatment.

Original languageEnglish (US)
Pages (from-to)A1308
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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