Ultrastructure and Ca++-ATPase activity of gap junctions under cryo-electron microscopic observation.

Abstract

In order to investigate the ultrastructure and Ca+ + -activated adenosine triphosphatase (Ca++-ATPase) activity of gap junctions under near-to-native conditions, frozen thin section and isolated gap junctons from rat liver or heart were examined under a 200 kV electron microscope equipped with a cryo-transfer system.The connexon arrangement observed from a face-on view in isolated, negatively stained gap junctions revealed quite a different pattern between cryo-and conventional images, although transverse views in frozen thin section showed no notable findings on gap junction structure. That is to say, whereas a well-ordered hexagonal connexon arrangement could be recognized in conventional liver gap junction images, connexons in cryo-images did not exhibit any regularity in their arrangements. Furthermore, the average diameter of connexons in cryo-images was a little bigger than that of the conventional images. The localization of Ca++-ATPase activity on isolated heart gap junctions also displayed a different appearance between cryo-and conventional images.These results suggest a structural change of isolated gap junctions during dispensable treatments, so the method of isolated gap junction observation under a cryo-electron microscope is considered to be very useful in detecting its near-tonative structure.Gap junctons are ultrastructurally composed of pairs of apposing plasma membrane connected with membrane proteins, called connexons (5, 10). The connexons are arranged in a hexagonal lattice in the plane of the membrane, and protrude into extracellular space to create a 2-3 nm-wide gap betwwen the membranes (4, 25). The connexon is itself an assembly of six identical transmembrane subunits (5, 33), pairs of which span both membrane bilayers and form a narrow continuous cell-to-cell aqueous pathway, a hydrophilic channel ca. 2 nm in diameter through the membrane (4). This generally well-accepted model of gap junction structure has been propsed