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Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

Meier S, Tavraz NN, Dürr KL, Friedrich T - J. Gen. Physiol. (2010)

Bottom Line: Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies.The leakage currents are prevented by aromatic amino acids at the carboxy terminus.Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany.

ABSTRACT
The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

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Reaction scheme and structural detail of the Na+/K+-ATPase. (A) Modified Post-Albers reaction cycle of the Na+/K+-ATPase. Upon intracellular binding of Na+ ions to the E1 conformation, a phosphointermediate with occluded Na+ ions, E1P(3Na+), is formed, and after a conformational change to E2P(3Na+), the Na+ ions dissociate to the extracellular space. Subsequently, two K+ ions bind from the extracellular side and become occluded, a process that stimulates dephosphorylation, and after a conformational change from E2 to E1, the K+ ions are intracellularly released. The gray box indicates the reaction sequence that can be studied by voltage pulses at high [Na+]ext and [K+]ext = 0 in TEVC experiments. (B) Structure of the Na+/K+-ATPase according to PDB structure entry 3B8E (Morth et al., 2007). Amino acids referred to in this work are indicated in ball-and-stick representation with numbering according to the human Na+/K+-ATPase α2 subunit. Helix M5 is depicted in yellow, the backbone of the carboxy terminus (V1014-EKETY-Y1020) is in red, and residues of a carboxy-terminal arginine cluster are in olive. Two Rb+ ions at the binding sites are shown as magenta spheres. Note that Arg1005 in the 3B8E structure (pig renal α1 subunit) corresponds to Tyr1009 in the human Na+/K+-ATPase α2 subunit.
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fig1: Reaction scheme and structural detail of the Na+/K+-ATPase. (A) Modified Post-Albers reaction cycle of the Na+/K+-ATPase. Upon intracellular binding of Na+ ions to the E1 conformation, a phosphointermediate with occluded Na+ ions, E1P(3Na+), is formed, and after a conformational change to E2P(3Na+), the Na+ ions dissociate to the extracellular space. Subsequently, two K+ ions bind from the extracellular side and become occluded, a process that stimulates dephosphorylation, and after a conformational change from E2 to E1, the K+ ions are intracellularly released. The gray box indicates the reaction sequence that can be studied by voltage pulses at high [Na+]ext and [K+]ext = 0 in TEVC experiments. (B) Structure of the Na+/K+-ATPase according to PDB structure entry 3B8E (Morth et al., 2007). Amino acids referred to in this work are indicated in ball-and-stick representation with numbering according to the human Na+/K+-ATPase α2 subunit. Helix M5 is depicted in yellow, the backbone of the carboxy terminus (V1014-EKETY-Y1020) is in red, and residues of a carboxy-terminal arginine cluster are in olive. Two Rb+ ions at the binding sites are shown as magenta spheres. Note that Arg1005 in the 3B8E structure (pig renal α1 subunit) corresponds to Tyr1009 in the human Na+/K+-ATPase α2 subunit.

Mentions: The Na+/K+-ATPase is an electrogenic ion pump, which exports three Na+ ions and imports two K+ ions at the expense of one ATP molecule. The reaction cycle of the Na+/K+-ATPase is commonly expressed as a sequence of reversible partial reactions known as the Post-Albers scheme (Fig. 1 A) (Albers, 1967; Post et al., 1972). The sequential translocation of Na+ and K+ ions requires strict cation specificity of the phosphorylation and dephosphorylation reactions, and the changes in the apparent affinities for the individual cation species are accompanied by alternating exposure of ion binding sites toward the intracellular and extracellular medium.


Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

Meier S, Tavraz NN, Dürr KL, Friedrich T - J. Gen. Physiol. (2010)

Reaction scheme and structural detail of the Na+/K+-ATPase. (A) Modified Post-Albers reaction cycle of the Na+/K+-ATPase. Upon intracellular binding of Na+ ions to the E1 conformation, a phosphointermediate with occluded Na+ ions, E1P(3Na+), is formed, and after a conformational change to E2P(3Na+), the Na+ ions dissociate to the extracellular space. Subsequently, two K+ ions bind from the extracellular side and become occluded, a process that stimulates dephosphorylation, and after a conformational change from E2 to E1, the K+ ions are intracellularly released. The gray box indicates the reaction sequence that can be studied by voltage pulses at high [Na+]ext and [K+]ext = 0 in TEVC experiments. (B) Structure of the Na+/K+-ATPase according to PDB structure entry 3B8E (Morth et al., 2007). Amino acids referred to in this work are indicated in ball-and-stick representation with numbering according to the human Na+/K+-ATPase α2 subunit. Helix M5 is depicted in yellow, the backbone of the carboxy terminus (V1014-EKETY-Y1020) is in red, and residues of a carboxy-terminal arginine cluster are in olive. Two Rb+ ions at the binding sites are shown as magenta spheres. Note that Arg1005 in the 3B8E structure (pig renal α1 subunit) corresponds to Tyr1009 in the human Na+/K+-ATPase α2 subunit.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812498&req=5

fig1: Reaction scheme and structural detail of the Na+/K+-ATPase. (A) Modified Post-Albers reaction cycle of the Na+/K+-ATPase. Upon intracellular binding of Na+ ions to the E1 conformation, a phosphointermediate with occluded Na+ ions, E1P(3Na+), is formed, and after a conformational change to E2P(3Na+), the Na+ ions dissociate to the extracellular space. Subsequently, two K+ ions bind from the extracellular side and become occluded, a process that stimulates dephosphorylation, and after a conformational change from E2 to E1, the K+ ions are intracellularly released. The gray box indicates the reaction sequence that can be studied by voltage pulses at high [Na+]ext and [K+]ext = 0 in TEVC experiments. (B) Structure of the Na+/K+-ATPase according to PDB structure entry 3B8E (Morth et al., 2007). Amino acids referred to in this work are indicated in ball-and-stick representation with numbering according to the human Na+/K+-ATPase α2 subunit. Helix M5 is depicted in yellow, the backbone of the carboxy terminus (V1014-EKETY-Y1020) is in red, and residues of a carboxy-terminal arginine cluster are in olive. Two Rb+ ions at the binding sites are shown as magenta spheres. Note that Arg1005 in the 3B8E structure (pig renal α1 subunit) corresponds to Tyr1009 in the human Na+/K+-ATPase α2 subunit.
Mentions: The Na+/K+-ATPase is an electrogenic ion pump, which exports three Na+ ions and imports two K+ ions at the expense of one ATP molecule. The reaction cycle of the Na+/K+-ATPase is commonly expressed as a sequence of reversible partial reactions known as the Post-Albers scheme (Fig. 1 A) (Albers, 1967; Post et al., 1972). The sequential translocation of Na+ and K+ ions requires strict cation specificity of the phosphorylation and dephosphorylation reactions, and the changes in the apparent affinities for the individual cation species are accompanied by alternating exposure of ion binding sites toward the intracellular and extracellular medium.

Bottom Line: Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies.The leakage currents are prevented by aromatic amino acids at the carboxy terminus.Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany.

ABSTRACT
The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

Show MeSH
Related in: MedlinePlus