Amino acid substitutions in the rat Na+,K+-ATPase alpha 2-subunit alter the cation regulation of pump current expressed in HeLa cells

1. To study the functional role of negatively charged amino acids (E327 and D925) located in the transmembrane region of the rat alpha 2-isoform 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 combination with a system for rapid solution changes. 2. Amino acid residues were replaced by glutamine (E327Q) or leucine (D925L) and were introduced into rat alpha 2* cDNA which encodes a ouabain-resistant isoform. These mutant enzymes were stably expressed in HeLa cells. The endogenous ouabain-sensitive HeLa cell Na+,K+-ATPase activity was selectively inhibited by 1 mu M ouabain present in both the growing media and the assay solution. 3. External K+- and internal Na+-dependent pump activation was observed in all cells expressing rat alpha 2*, E327Q or D925L; however, the apparent affinities were significantly reduced by the mutations. 4. In E327Q, the activation of pump current was slightly slower than for rat alpha 2*, whereas the deactivation rate was faster. In contrast, D925L produced pump current having dramatically slower activation and deactivation kinetics. 5. These results indicate that these negatively charged amino acids (E327 and D925) are important in cation-induced conformational changes of the protein, which are intermediate steps in the pump mechanism.