The current produced by the E779A mutant rat Na+/K+ pump alpha 1-subunit expressed in HEK 293 cells

The current (I-p) generated by the wild-type or the glutamate (E) 779 alani ne (A) mutant of the rat Na+/K+ pump alpha1-subunit expressed in HEK 293 ce lls was studied at 35 degreesC by means of whole-cell recording in Na+-free and Na+-containing solution. Glutamate 779 is located in the fifth transme mbrane domain of the alpha -subunit of the Na+/K+-ATPase. Compared with the wild-type, the E779A mutant exhibited an apparent K-0(+)-affinity decrease d by a factor of 3-4 both in Na+-free and in Na+-containing media. The comp etition of Na-0(+) and K-0(+) for cation binding sites of the pump remained unchanged. Similarly, in Na+-free solution the shape of the I-p-V curves f or various external K+-concentrations ([K+](0)) was essentially the same. H owever, in Na+-containing solutions the shape of I-p-V curves from cells ex pressing the mutant of the rat alpha1-subunit clearly differed from the sha pe observed in cells expressing the wild-type, but voltage dependence of th e pump current persisted. A prominent Na-0(+)-activated, electrogenic Na+-t ransport 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-0 (+) affinity and a profound, Na-0(+)-dependent alteration in the electrogen icity of the mutant pump expressed in HEK 293 cells. (C) 2000 Elsevier Scie nce B.V. All rights reserved.