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 α₁ 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 α₁ 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 ~7.5 Å revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an α-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.