Electron microscopic image analysis of cardiac gap junction membrane crystals.
Cardiac gap junctions play an important functional role in the myocardium by electrically coupling adjacent cells, thereby providing a low resistance pathway for cell-to-cell propagation of the action potential. Two-dimensional crystallization of biochemically isolated rat ventricular gap junctions has been accomplished by an in situ method in which membrane suspensions are sequentially dialyzed against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are partially extracted without solubilizing the protein, and the increased protein concentration facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Projection density maps show that the connexons in cardiac gap junctions are formed by a hexameric cluster of alpha 1 connexin subunits. Protease cleavage of alpha 1 connexin from 43 to 30 kDa releases approximately 13kDa from the carboxy-tail, and the projection density maps are not significantly altered. Uranyl acetate stain penetrates the ion channel, whereas phosphotungstic acid is preferentially deposited over the lipid regions. This differential staining can be used to selectively probe the central channel of the connexon and the interface between the connexon and the lipid. The hexameric design of alpha 1 connexons appears to be a recurring quaternary motif for the multigene family of gap junction proteins.
Yeager
M
M
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
D000595:Amino Acid Sequence; D000818:Animals; D002450:Cell Communication; D017630:Connexins / Q000737:chemistry / Q000648:ultrastructure*; D003460:Crystallization; D004577:Electron Probe Microanalysis; D017629:Gap Junctions / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D007473:Ion Channels / Q000648:ultrastructure*; D008099:Liver / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009206:Myocardium / Q000648:ultrastructure*; D017434:Protein Structure, Tertiary; D051381:Rats
D017630:Connexins; D007473:Ion Channels