MutagPDPsis of Ghr2' in the 4^th transmembrane segment of the rt-subuiiil of Na.K ATPase was performed to distinguish effects of mutations on the V Y.-i conformational transitions from direct interference with cation binding and nrrluriioTi. Substitutions of GIu³²⁷ for Gin reduces Na,K-ATPase activity and Na dependent phosphorylation to about 1/3 and these activities are eliminated in Glu³²⁷ Asp. The mutations do not alter high affinity binding of pH] AT P or pH]-ouabain and chyrnotryptic digestion yields normal cleavage prodiu.ts a.s an indication that the structure of rytoplasmic and extracelluiar ciornaina are preserved. The mutations abolish high affinity occlusion of ²⁰⁴T1⁺ ions and the affinities of K ⁺-ions for displacement of [3H]-ATP arc reduced in parallel. Neither Na⁺ nor K⁺ (10 mM) are able to alter the conformationai -tate of the o subunit of Glu327Gln as monitored with chymolrypsin. while transition to the E|-form or £2-form are observed after binding of AT P 01 ouabain. Interference with occiusion of K+ in the F-f-Kj-form and of Na" in the- KiP[3Naj-form, may therefore bo due to removal of groups coordinating K⁺ or Na⁺ in the occiusion cavities.