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 Na(+)/K(+) pump and its molecular expression in L8 rat muscle cells. Pump properties were measured by [(3)H]ouabain binding and (86)Rb uptake. Scatchard plot analysis of specific ouabain binding indicated the presence of a single family of binding sites with a B(max) of approximately 135 fmol/ mg P and a K(D) of 3.3 x 10(-8). (86)Rb 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 alpha(1) and beta(1) protein and mRNA isoforms were expressed in myoblasts and in myotubes, while 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 employing a vector expressing an avian high affinity alpha 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 alpha 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 alpha 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 alpha subunits hybridize to form functional pump complexes.
Sharabani-Yosef
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Faculty of Life Sciences, Gonda-Goldschmeid Center, Bar-Ilan University, Ramat-Gan, Israel.
Bak
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Nir
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Sampson
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D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D008564:Membrane Potentials / Q000502:physiology; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis; D021122:Protein Subunits; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012413:Rubidium / Q000493:pharmacokinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium