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

We have characterized the physiological and biochemical properties of the N a+/ K+ pump and its molecular expression in L8 rat muscle cells. Pump prope rties were measured by [H-3]ouabain binding and Rb-86 uptake. Scatchard plo t analysis of specific ouabain binding indicated the presence of a single f amily of binding sites with a B-max of similar to 135 fmol/ mg P and a K-D of 3.3 x 10(-8). Rb-86 uptake due to specific pump activity was found to be 20% of the total in L8 cells. The results indicated lower affinity of L8 c ells for ouabain and lower activity of the pump than that reported for chic k or rat skeletal muscle in primary culture. Both the alpha (1) and beta (1 ) protein and mRNA isoforms were expressed in myoblasts and in myotubes, wh ile the alpha (2), alpha (3), and beta (2) isoforms were not detectable. We attempted to overcome low physiological expression of the Na+/K+ pump by e mploying a vector expressing an avian high affinity alpha subunit. This all owed identification of the transfected subunit separate from that endogenou sly expressed in L8 cells. Successful transfection into L8 myoblasts and my otubes was recognized by anti-avian a subunit monoclonal antibodies. Fusion index, Na+/K+ pump activity, and the level of the transmembrane resting po tential were all significantly greater in transfected L8 (tL8) cells than i n non-tL8. The total amount of alpha subunit (avian and rat) in tL8 cells w as greater than that (only rat) in non-tL8 cells. This relatively high abun dance of the Na+/K+ pump in transfected cells may indicate that avian and r at a subunits hybridize to form functional pump complexes. J. Cell. Physiol . 187: 365-373, 2001. (C) 2001 Wiley-Liss, Inc.