Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity.
The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.
Imagawa
Toshiaki
T
Biochemistry, Division of Chemistry, Graduate School of Science, Hokkaido University, Nishi, Sapporo 060-0810, Japan. toshi@sci.hokudai.ac.jp
Yamamoto
Tetsuya
T
Kaya
Shunji
S
Sakaguchi
Kazuyasu
K
Taniguchi
Kazuya
K
D009597:4-Nitrophenylphosphatase / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D010100:Oxygen / Q000737:chemistry; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*; D014162:Transfection; D014633:Valine / Q000737:chemistry*
D002412:Cations; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D049452:Green Fluorescent Proteins; D013912:Threonine; D018698:Glutamic Acid; D012965:Sodium Chloride; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D014633:Valine; D011188:Potassium; D010100:Oxygen