(Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

Open Access
Publisher Website
Google Scholar
Abstract
Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of Na and K [Na,K]-ATPase on the surface of tissue-cultured chick skeletal muscle. At this level of resolution, the (Na,K)-ATPase molecules were evidently distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a 2nd antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescein-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of 3 other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. The (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-ptching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that was a 1:1 correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. The intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to .apprx. 400 particles/.mu.m2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP. A hypothesis is advanced to account for the reported difference in particle density observed in freeze-fracture replicas of normal and dystrophic skeletal muscle.