The current produced by the E779A mutant rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells.
The current (I(p)) generated by the wild-type or the glutamate (E) 779 alanine (A) mutant of the rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells was studied at 35 degrees C by means of whole-cell recording in Na(+)-free and Na(+)-containing solution. Glutamate 779 is located in the fifth transmembrane domain of the alpha-subunit of the Na(+)/K(+)-ATPase. Compared with the wild-type, the E779A mutant exhibited an apparent K(+)(o)-affinity decreased by a factor of 3-4 both in Na(+)-free and in Na(+)-containing media. The competition of Na(+)(o) and K(+)(o) for cation binding sites of the pump remained unchanged. Similarly, in Na(+)-free solution the shape of the I(p)-V curves for various external K(+)-concentrations ([K(+)](o)) was essentially the same. However, in Na(+)-containing solutions the shape of I(p)-V curves from cells expressing the mutant of the rat alpha1-subunit clearly differed from the shape observed in cells expressing the wild-type, but voltage dependence of the pump current persisted. A prominent Na(+)(o)-activated, electrogenic Na(+)-transport mediated by the pump, displaying little voltage dependence in the potential range tested (-80 to +60 mV), was present in the cells expressing the E779A mutant pump. The data suggest that exchanging E779 for A in the rat Na(+)/K(+) pump alpha1-subunit causes a modest decrease in the apparent K(+)(o) affinity and a profound, Na(+)(o)-dependent alteration in the electrogenicity of the mutant pump expressed in HEK 293 cells.
Zillikens
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Arbeitsgruppe Muskelphysiologie, Ruhr-Universität Bochum, Germany.
Gisselmann
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G
Glitsch
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HG
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D004073:Digitoxigenin; D004563:Electrochemistry; D007477:Ions / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011188:Potassium / Q000737:chemistry; D051381:Rats; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012996:Solutions; D014162:Transfection
D003470:Culture Media; D007477:Ions; D012996:Solutions; D004073:Digitoxigenin; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium