Expression, two-dimensional crystallization, and electron cryo-crystallography of recombinant gap junction membrane channels.
We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.
Unger
V M
VM
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Kumar
N M
NM
Gilula
N B
NB
Yeager
M
M
D000818:Animals; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000648:ultrastructure*; D006224:Cricetinae; D020285:Cryoelectron Microscopy; D003460:Crystallization; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D007473:Ion Channels / Q000648:ultrastructure*; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000648:ultrastructure; D014162:Transfection
D018031:Connexin 43; D007473:Ion Channels; D011994:Recombinant Proteins