The GLUT4 glucose transporter and the α2subunit of the Na+,K+‐ATPase do not localize to the same intracellular vesicles in rat skeletal muscle

The GLUT4 glucose transporter and the α2subunit of the Na+,K+‐ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co‐segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single‐ and double‐labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K+‐ATPase α2subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The α2subunit of the Na+,K+‐ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double‐labeling of GLUT4 and Na+,K+‐ATPase α2subunit revealed that less than 6% of the two proteins co‐localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4‐containing membranes from muscle homogenates, in which the α2subunit of the Na+,K+‐ATPase was found only at the same extent as the α1subunit of the enzyme, a protein exclusively present at the plasma membrane.