Alanine-scanning mutagenesis of the sixth transmembrane segment of gastric H+,K+-ATPase alpha-subunit.
The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H(+),K(+)-ATPase alpha-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H(+),K(+)-ATPase; overall K(+)-stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K(+)-ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K(+), whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the alpha-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K(+)-dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K(+)-ATPase activity accompanying the decrease in the rate of phosphorylation.
Asano
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S
Molecular Genetics Research Center and the Faculty of Pharmaceutical Sciences of Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. shinji@ms.toyama-mpu.ac.jp
Io
T
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Kimura
T
T
Sakamoto
S
S
Takeguchi
N
N
D000409:Alanine; D000595:Amino Acid Sequence; D002460:Cell Line; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D021122:Protein Subunits; D013270:Stomach / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013696:Temperature
D021122:Protein Subunits; D017506:H(+)-K(+)-Exchanging ATPase; D000409:Alanine