Involvement in K+ access of Leu318 at the extracellular domain flanking M3 and M4 of the Na+,K+-ATPase alpha-subunit |
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Authors: | Eguchi Hiroshi Takeda Kazuo Schwarz Wolfgang Shirahata Akira Kawamura Masaru |
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Institution: | Department of Cell Biology, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan. |
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Abstract: | The effect of point mutation in the sequence 316TWLE319, which occurs in the extracellular loop flanking the third (M3) and the fourth (M4) transmembrane segment (L3/4) of the Na+,K+-ATPase alpha-subunit, was examined. Mutation of Glu319 to Asp yielded an enzyme with full activity, whereas substituting Glu319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr316 to Leu318 (by E-scanning) in the mutant construct with Glu319 already mutated to Gln. The activity that had been reduced to 60% by the mutation of Glu319 to Gln was restored upon the introduction of a negative charge by E-scanning. When Leu318 was replaced by Glu in a series of scanning experiments, the K+ sensitivity of the ATPase activity was lowered. The lowering of K+ sensitivity was further demonstrated when a mutation of Leu318 to Glu was introduced into the wild-type enzyme. Furthermore, mutants with Leu318 to Gln, Arg, and Phe displayed lower K+ sensitivity similar to that of Leu318 to Glu mutant. Leu318 may be in access path for K+, and any substitution at this position may interfere with access of K+ from outside the cell. |
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Keywords: | Na+ K+-ATPase K+ Access path Extracellular domain Mutagenesis |
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