title: Involvement in K+ access of Leu318 at the extracellular domain flanking M3 and M4 of the Na+,K+-ATPase alpha-subunit.
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.
authors.affiliation: Department of Cell Biology, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Eguchi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takeda
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schwarz
authors.forename: Akira
authors.initials: A
authors.lastname: Shirahata
authors.forename: Masaru
authors.initials: M
authors.lastname: Kawamura
mesh_terms: D000818:Animals; D007930:Leucine / Q000737:chemistry*; D016296:Mutagenesis; D017354:Point Mutation; D011188:Potassium / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
chemical_list: D000254:Sodium-Potassium-Exchanging ATPase; D007930:Leucine; D011188:Potassium