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

Mads Toustrup-Jensen,Bente Vilsen

doi:10.1074/jbc.m305521200

Abstract

Mutations Ile279 --> Ala, Ile283 --> Ala, Glu284 --> Ala, His285 --> Ala, His285 --> Lys, His285 --> Glu, Phe286 --> Ala, and His288 --> Ala in transmembrane helix M3 of the Na+,K(+)-ATPase were studied. Except for His285 --> Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K(+)-transport cycle. In Ile279 --> Ala and Ile283 --> Ala, the E1 form accumulated, whereas in His285 --> Lys and His285 --> Glu, E1P accumulated. Phe286 --> Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P --> E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1-E2 and E1P-E2P conformational changes. Because the E1-E2 and E1P-E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na(+)-activated ATPase activity in the absence of K+ ("Na(+)-ATPase activity"). Ile279 --> Ala, Ile283 --> Ala, and His288 --> Ala showed reduced Na+ affinity of the E1 form. The rate of Na(+)-activated phosphorylation from ATP was reduced in Ile279 --> Ala and Ile283 --> Ala, and these mutants showed evidence similar to Glu329 --> Gln of destabilization of the Na(+)-occluded state.

Highlights

The Naϩ,Kϩ-ATPase1 present in the plasma membranes of all animal cells utilizes the energy derived from hydrolysis of ATP to drive transport of Naϩ and Kϩ ions in opposite directions against their electrochemical gradients [1]
Virtually nothing is known about the determinants of the specific cation binding properties and the pathways of ion migration to and from the binding pocket. Another important issue concerns the mechanism of energy transduction: how ATP hydrolysis is linked to ion transport? This must involve long range interaction between the cytoplasmic domains and the cation coordinating residues located some 40 Å away in the transmembrane segments
Titration of the ouabain concentration dependence of Naϩ,KϩATPase activity revealed that the K0.5 value for ouabain inhibition displayed by the mutants Glu284 3 Ala and His288 3 Ala was very similar to that of the wild type, whereas the K0.5 value was increased ϳ4-fold in Ile279 3 Ala, His285 3 Glu, and His285 3 Lys, and ϳ6-fold in Ile283 3 Ala and Phe286 3 Ala relative to the wild type (Table I)

Summary

Introduction

The Naϩ,Kϩ-ATPase present in the plasma membranes of all animal cells utilizes the energy derived from hydrolysis of ATP to drive transport of Naϩ and Kϩ ions in opposite directions against their electrochemical gradients [1]. From mutational studies it emerges that the residues of Naϩ,Kϩ-ATPase most likely to be involved in Naϩ and Kϩ binding are located in the transmembrane segments M4, M5, and M6 Role of M3 Mutations in Naϩ,Kϩ-ATPase apparent affinity for Naϩ in the E1 form, whereas others allowed Naϩ to substitute for Kϩ at the extracellularly facing sites of the E2P form, leading to an unusually high Naϩ-induced ATPase activity in the absence of Kϩ [29] The latter effect has been observed for mutation of certain residues located in or close to the cation-binding pocket

Results

Discussion

Conclusion