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-hydra ted, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal trun cation 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 analy sis of a membrane protein that has been accomplished using microgram amount s of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously as sembled gap junction plaques. Detergent. treatment with Tween 20 and 1,2-di heptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-d imensional density (3D) map with an in-plane resolution of similar to 7.5 A ngstrom revealed that each hexameric connexon was formed by 24 closely pack ed rods of density, consistent with an alpha-helical conformation for the f our transmembrane domains of each connexin subunit. In the extracellular ga p the aqueous channel was bounded by a continuous wall of protein that form ed a tight electrical and chemical seal to exclude exchange of substances w ith the extracellular milieu (C) 1999 Academic Press.