AMINO-ACID SUBSTITUTIONS IN THE RAT NA-ATPASE ALPHA-2-SUBUNIT ALTER THE CATION REGULATION OF PUMP CURRENT EXPRESSED IN HELA-CELLS(,K+)

1. To study the functional role of negatively charged amino acids (E32 7 and D925) located in the transmembrane region of the rat alpha 2-iso form of the Na+,K+-ATPase (rat alpha 2) in ion transport, the effects of mutations on external K+ dependence and internal Na+ dependence of pump currents were assessed by the patch-clamp technique in combinati on with a system for rapid solution changes. 2. Amino acid residues we re replaced by glutamine (E327Q) or leucine (D925L) and were introduce d into rat alpha 2 cDNA which encodes a ouabain-resistant isoform. Th ese mutant enzymes were stably expressed in HeLa cells. The endogenous ouabain-sensitive HeLa cell Na+,K+-ATPase activity was selectively in hibited by 1 mu M ouabain present in both the growing media and the as say solution. 3. External K+- and internal Na+-dependent pump activati on was observed in all cells expressing rat alpha 2, E327Q or D925L; however, the apparent affinities were significantly reduced by the mut ations. 4. In E327Q, the activation of pump current was slightly slowe r than for rat alpha 2, whereas the deactivation rate was faster. In contrast, D925L produced pump current having dramatically slower activ ation and deactivation kinetics.5. These results indicate that these n egatively charged amino acids (E327 and D925) are important in cation- induced conformational changes of the protein, which are intermediate steps in the pump mechanism.