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

Abstract

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 alpha1-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 alpha1-subunit may be found among the fibers. We utilized two well-characterized specific antibodies for the alpha1-subunit, namely the McK1 and alpha6F, to determine, by immunofluorescence, if the alpha1-subunit in rat skeletal muscle fiber is differentially phosphorylated. McK1 has the unique property that its binding to the alpha1-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 alpha6F antibody showed that the alpha1-subunit is expressed in all fibers. Dephosphorylation of the tissue sections by phosphatase partially restored immunostaining of the alpha1-subunit by McK1. Fiber typing results showed that, in red gastrocnemius, those fibers stained positive for alpha1-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 alpha1-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 alpha1-subunit, in addition to the previously demonstrated increase in the levels of expression of the subunit.

The α1 isoform of the Na+/K+ ATPase is up-regulated in dedifferentiated progenitor cells that mediate lens and retina regeneration in adult newts

“…We used a monoclonal antibody (α6F) that is specific for this isoform. This antibody has been widely employed to distinguish Na + /K + ATPase α1 from the other isoforms by immunohistochemistry and Western blot in different tissues, including the retina, of various animal models (Arteaga et al, 2004; Drummond et al, 1998; Hlivko et al, 2006; Mobasheri et al, 2003; Wetzel et al, 1999; Zhang and Ng, 2007). Moreover, the epitope that is recognized by this antibody includes one of the polypeptide sequences that we had identified in our mass spectrometry analysis (highlighted in figure 1A and red in supplementary figure 1).…”

“…α6F, an antibody that is specific for the α1 isoform of the Na + /K + ATPase (Arteaga et al, 2004; Drummond et al, 1998; Hlivko et al, 2006; Mobasheri et al, 2003; Wetzel et al, 1999; Zhang and Ng, 2007) was obtained from a hybridoma cell culture to be used for Western blot and immunohistochemical studies. Briefly, α6F hybridoma cells developed by Dr. D.M.…”

Impaired AQP2 trafficking in Fxyd1 knockout mice: A role for FXYD1 in regulated vesicular transport

“…Fresh-frozen cryosections (10 μm) from wild type or Fxyd1 -/- kidneys were incubated with 100 μl lambda-phosphatase reaction buffer (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 0.1 mM EGTA, 2 mM dithiothreitol and 0.01% Brij 35) for 5 min at room temperature [ 42 ]. The incubation solution was changed to a dephosphorylation solution (100 μl) containing 400 U of lambda protein phosphatase (λPP) (New England Biolabs, Ipswich, MA), 2 mM MnCl 2 , lambda-protein phosphatase reaction buffer, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO).…”

Effects of Age on Na+,K+-ATPase Expression in Human and Rodent Skeletal Muscle

“…From the above findings and also the reduction of α 2 β 2 in aged isoforms in rat, it seems reasonable to suggest in aged rats, that the increased abundance of the α 1 β 1 isoforms may be compensatory for these losses. It was also shown in red gastrocnemius muscle, that α 1 was less phosphorylated in aged rats (Zhang and Ng, 2007 ), although the functional implications of this are not yet understood.…”