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 200kV 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 gapjunction 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-to-native structure.