Fiber specific differential phosphorylation of the α1-subunit of the Na+,K+-ATPase in rat skeletal muscle: the effect of aging

In skeletal muscle, the Na+,K+-ATPase maintains the Na+ and K+ gradients and modulates contractile functions. The different fibers of the skeletal muscle possess diverse properties and functions, and thus, the demands for the Na-pump activity might be different. Because phosphorylation of the α1-subunit of the Na+,K+-ATPase appears to serve a regulatory role in the activity of Na+,K+-ATPase, we postulated that a difference in the phosphorylation of the α1-subunit may be found among the fibers. We utilized two well-characterized specific antibodies for the α1-subunit, namely the McK1 and α6F, to determine, by immunofluorescence, if the α1-subunit in rat skeletal muscle fiber is differentially phosphorylated. McK1 has the unique property that its binding to the α1-subunit is greatly reduced when Ser-18 is phosphorylated. Our data show that, in red gastrocnemius muscle, only a small number of the fibers were stained on the sarcolemmal membrane by McK1, while other fibers were almost completely devoid of any staining. By contrast, the staining pattern by McK1 in the white gastrocnemius muscle was mostly uniform. Immunostaining of serial sections using the α6F antibody showed that the α1-subunit is expressed in all fibers. Dephosphorylation of the tissue sections by phosphatase partially restored immunostaining of the α1-subunit by McK1. Fiber typing results showed that, in red gastrocnemius, those fibers stained positive for α1-subunit by McK1 are the Type I fibers, whereas those stained negative are the Type IIA, IID, and IIB fibers. With age, the number of fibers in red gastrocnemius stained positive for McK1 increased markedly in 30-month old rats compared to 6-month old rats. In conclusion, our result suggests that, in rats, the α1-subunit of the Na+,K+-ATPase is differentially phosphorylated in the fibers of the red gastrocnemius muscle. Furthermore, advanced age is associated with an apparent decrease in the phosphorylation of the α1-subunit, in addition to the previously demonstrated increase in the levels of expression of the subunit.