Item

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 α-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.