Mutation of Aspartate 804 of Na⁺,K⁺-ATPase Modifies the Cation Binding Pocket and Thereby Generates a High Na⁺-ATPase Activity

Abstract

A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.

Chimeras of X+,K+-ATPases

“…This effect has been observed previously (10) for the Na ϩ ,K ϩ -ATPase mutant D804A. It might be that the presence of the chimera in the E 1 conformation is sufficient for activating the phosphorylation process and thus initiates the ATPase reaction.…”

“…ATPase Activity Assay-The ATPase activity was determined with a radiochemical method (10). For this purpose Sf9 membranes were added to 100 l of medium, which contained under standard conditions 50 mM Tris-acetic acid (pH 7.0), 0.2 mM EDTA, 0.1 mM EGTA, 1 mM Tris-N 3 , 1.3 mM MgCl 2 , 10 mM KCl, 100 mM NaCl, and 100 M [␥- 3-6.…”

“…This difference is due to the much faster rate of the E 2 K 3 E 1 K conversion in the latter enzyme (6,38). It has been shown that four negatively charged residues present in transmembrane segments 4 -6 play a role in cation-activated reactions of both ATPases (7)(8)(9)(10)(11)(12)(13). With the exception of a single residue in M6…”

Functional Consequences of Alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal Part of Transmembrane Helix M3 of the Na+,K+-ATPase

“…The enzyme was equilibrated with K ϩ in the absence of Na ϩ and ATP. Oligomycin was then added to block dephosphorylation, and subsequently the phosphorylation reaction was initiated by diluting the enzyme 10-fold in a solution containing [␥- 32 P]ATP and Na ϩ , and the time course of phosphoenzyme formation was followed. The reaction sequence leading to phosphorylation of E 2 (K 2 ) proceeds through the steps E 2 (K 2 ) 3 E 1 3 E 1 Na 3 3 E 1 P(Na 3 ), where the release of occluded K ϩ is rate-limiting.…”

A missense variant of the ATP1A2 gene is associated with a novel phenotype of progressive sensorineural hearing loss associated with migraine

“…The ouabain-sensitive ATPase activity was determined using the radiochemical method of Koenderink et al 13 For this purpose, 0.6-5 mg of Sf9 membranes were added to 100 ml of medium, which contained 10-200 mM [g-32 P]-ATP (specific activity 20-100 mCi/mmol, Amersham Pharmacia Biotech, Buckinghamshire, UK), 1.2 mM MgCl 2 , 0.2 mM EGTA, 0.1 mM EDTA, 0.1 mM ouabain, 1 mM NaN 3 , 25 mM Tris-HCl (pH 7.0) and various concentrations of KCl and NaCl in the presence and absence of 0.1 mM ouabain. After incubation for 30 min at 37 1C, the reaction was stopped by adding 500 ml 10% (w/v) charcoal in 6% (v/v) trichloroacetic acid.…”