Substitution of Glutamic 779 with Alanine in the Na,K-ATPase α Subunit Removes Voltage Dependence of Ion Transport

Abstract

The effects of changing Glu-779, located in the fifth transmembrane segment of the Na,K-ATPase ␣ subunit, on the phosphorylation characteristics and ion transport properties of the enzyme were investigated. HeLa cells were transfected with cDNA coding the E779A substitution in an ouabain-resistant sheep ␣1 subunit (RD). Steady state phosphorylation stimulated by Na ؉ concentrations less than 20 mM or by imidazole were similar for RD and E779A enzymes, an indication that phosphorylation and Na ؉ occlusion were not altered by this mutation. With E779A enzyme, higher Na ؉ concentrations reduced the level of phosphoenzyme and stimulated Na-ATPase activity in the absence of K ؉ . These effects were a consequence of Na ؉ increasing the rate of protein dephosphorylation. In voltage-clamped HeLa cells expressing E779A enzyme, a prominent electrogenic Na ؉ -Na ؉ exchange was observed in the absence of extracellular K ؉ . Thus, increased Na-ATPase activity and Na ؉ -dependent dephosphorylation result from Na ؉ acting as a K ؉ congener with low affinity at extracellular binding sites. These data suggest that E779A does not directly participate in ion binding but does affect the connection between extracellular ion binding and intracellular enzyme dephosphorylation. In cells expressing control RD enzyme, Na,K-pump current was dependent on membrane potential and extracellular K ؉ concentration. However, Na,K-pump current in cells expressing E779A enzyme was voltage independent at all extracellular K ؉ tested. These results indicate that Glu-779 may be part of the access channel determining the voltage dependence of ion transport by the Na,K-ATPase.

Characterization of a Thermophilic P-type Ag+/Cu+-ATPase from the ExtremophileArchaeoglobus fulgidus

“…The phosphorylation of the aspartyl group in the consensus sequence DKTGT is a fundamental characteristic within this mechanism. The participation of specific transmembrane segments (TMs) in cation binding and transport has also been established for the better characterized members of this family, the Na,K-ATPase and SR Ca-ATPase (5)(6)(7)(8). Recently, the structure of the SR Ca-ATPase (2.6-Å resolution) was reported (9).…”

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

“…Apart from the requirement of molecular structure, there is an obvious need to carry out similar biophysical studies with the mutated pumps as have been done with the wild-type enzyme. In one case a mutation, E779A, has been suggested to affect the extracellular ion-well [7].…”

Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases

“…However, the most parsimonious model to rationalize our findings is one in which these four amino acids participate in metal coordination and consequently yield mutants unable to bind Cu ϩ . The position of putative Cu ϩ binding residues in the center of H6, H7, and H8 has remarkable similarity with the cation binding sites in the Na,K-ATPase and Ca-ATPase (32)(33)(34)(35)(36)(37)(38)(39)(40). In both cases, amino acids in the central portion of the TMs flanking the large cytoplasmic loop contribute side chain atoms for metal coordination.…”

“…A similar role has been indicated for transmembrane His and carboxylic amino acids in the case of NixA-type Ni 2ϩ permeases (29,30) and the DMT1 Fe 2ϩ transporters (31). In contrast, the transmembrane cation binding sites have been well described for the Ca-ATPase and Na,K-ATPase, extensively characterized P IItype cation pumps (32)(33)(34)(35)(36)(37)(38). Moreover, crystal structures of the cation-bound Ca-ATPase are available (39,40).…”