Alanine scanning mutagenesis of oxygen-containing amino acids in the transmembrane region of the Na,K-ATPase

Oxygen-containing amino acids in the transmembrane region of the Na,K-ATPas e alpha subunit were studied to identify residues involved in Na+ and/or K coordination by the enzyme. Conserved residues located in the polar face o f transmembrane helices were selected using helical wheel and topological m odels of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha 1 isoform and expressed in HeLa cell s. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of hete rologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, S er140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu8 18, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those c arrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were intr oduced into the wild-type alpha 1 sheep isoform and expressed in mouse NIH3 T3 cells where [H-3]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. E xpression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at th e plasma membrane was significantly lower than that of the wild-type isofor m. Thus, these substitutions appear to impair the maturation process or tar geting of the enzyme to the plasma membrane, but not cation-enzyme interact ions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-c ontaining residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme fu nction. Consequently, other chemical groups, aromatic rings or backbone car bonyls, should be considered in models of cation-binding sites. (C) 1999 Ac ademic Press.