Na+/K+ pump expression in the L8 rat myogenic cell line: Effects of heterologous α subunit transfection

Orna Sharabani-Yosef, Asia Bak, Uri Nir, Sanford R. Sampson

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4 Scopus citations


We have characterized the physiological and biochemical properties of the Na+/K+ pump and its molecular expression in L8 rat muscle cells. Pump properties were measured by [3H]ouabain binding and 86Rb uptake. Scatchard plot analysis of specific ouabain binding indicated the presence of a single family of binding sites with a Bmax of ∼135 fmol/mg P and a KD of 3.3 × 10-8. 86Rb uptake due to specific pump activity was found to be 20% of the total in L8 cells. The results indicated lower affinity of L8 cells for ouabain and lower activity of the pump than that reported for chick or rat skeletal muscle in primary culture. Both the α1 and β1 protein and mRNA isoforms were expressed in myoblasts and in myotubes, while the α2, α3, and β2 isoforms were not detectable. We attempted to overcome low physiological expression of the Na+/K+ pump by employing a vector expressing an avian high affinity α subunit. This allowed identification of the transfected subunit separate from that endogenously expressed in L8 cells. Successful transfection into L8 myoblasts and myotubes was recognized by anti-avian α subunit monoclonal antibodies. Fusion index, Na+/K+ pump activity, and the level of the transmembrane resting potential were all significantly greater in transfected L8 (tL8) cells than in non-tL8. The total amount of α subunit (avian and rat) in tL8 cells was greater than that (only rat) in non-tL8 cells. This relatively high abundance of the Na+/K+ pump in transfected cells may indicate that avian and rat α subunits hybridize to form functional pump complexes.

Original languageEnglish
Pages (from-to)365-373
Number of pages9
JournalJournal of Cellular Physiology
Issue number3
StatePublished - Jun 2001


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