The effect of point mutation in the sequence (TWLE319)-T-316, 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 Glu(319) to Asp yielded an enzyme with full activity, whereas Substituting Glu 319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr(316) to Leu(318) (by E-scanning) in the Mutant construct with Glu(319) already mutated to Gin. 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 Leu(318) was replaced by Gin 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 Leu(318) to Glu was introduced into the wild-type enzyme. Furthermore, Mutants with Leu(318) to Gin, Arg, and Phe displayed lower K+ sensitivity similar to that of Leu(318) to Glu mutant. Leu(318) may be in access path for K+, and any Substitution at this position may interfere with access of K+ from outside the cell. (c) 2005 Elsevier Inc. All rights reserved.