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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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Voltage and [K+]ext dependence of stationary currents at [Na+]ext = 100 mM. (A) I-V curves of normalized ouabain-sensitive K+-dependent currents NaIxK(ouab) of the Na+/K+-ATPase ΔYY deletion construct measured at 100 mM [Na+]ext and [K+]ext as indicated. (B) I-V curves of normalized K+-induced difference currents NaIxK (at 100 mM [Na+]ext) of the ΔYY construct. (C) Voltage-dependent NaK0.5(K+ext) values of the ΔYY construct from fits of a Hill function to the NaIxK currents in B at each membrane potential. The minimal NaK0.5(K+ext) was 1.02 ± 0.06 mM at −80 mV. The dashed line delineates the corresponding WT data from E for comparison. (D) I-V curves of normalized NaIxK difference currents for ATP1A2 WT with [K+]ext as indicated. (E) Voltage-dependent NaK0.5(K+ext) values for ATP1A2 WT from fits of a Hill function to the NaIxK currents in D at each membrane potential. The minimal NaK0.5(K+ext) was 1.10 ± 0.05 mM at 0 mV. Data for both WT and ΔYY are means ± SE from 14 cells of three oocyte batches. (F) I-V curves of normalized NaI10K(ouab) and NaI0K(ouab) currents of ATP1A2 WT and the ΔYY construct at pHext 7.4 ([Na+]ext = 100 mM). Data are means ± SE from six (WT) and seven (ΔYY) cells from two batches.
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fig2: Voltage and [K+]ext dependence of stationary currents at [Na+]ext = 100 mM. (A) I-V curves of normalized ouabain-sensitive K+-dependent currents NaIxK(ouab) of the Na+/K+-ATPase ΔYY deletion construct measured at 100 mM [Na+]ext and [K+]ext as indicated. (B) I-V curves of normalized K+-induced difference currents NaIxK (at 100 mM [Na+]ext) of the ΔYY construct. (C) Voltage-dependent NaK0.5(K+ext) values of the ΔYY construct from fits of a Hill function to the NaIxK currents in B at each membrane potential. The minimal NaK0.5(K+ext) was 1.02 ± 0.06 mM at −80 mV. The dashed line delineates the corresponding WT data from E for comparison. (D) I-V curves of normalized NaIxK difference currents for ATP1A2 WT with [K+]ext as indicated. (E) Voltage-dependent NaK0.5(K+ext) values for ATP1A2 WT from fits of a Hill function to the NaIxK currents in D at each membrane potential. The minimal NaK0.5(K+ext) was 1.10 ± 0.05 mM at 0 mV. Data for both WT and ΔYY are means ± SE from 14 cells of three oocyte batches. (F) I-V curves of normalized NaI10K(ouab) and NaI0K(ouab) currents of ATP1A2 WT and the ΔYY construct at pHext 7.4 ([Na+]ext = 100 mM). Data are means ± SE from six (WT) and seven (ΔYY) cells from two batches.

Mentions: Upon expression in Xenopus laevis oocytes, TEVC experiments were performed in Na+-containing buffers ([Na+]ext = 100 mM) to measure the K+ext-induced ouabain-sensitive pump currents of wild-type (WT) human α2/β1 Na+/K+-ATPase and of the ΔYY construct, in which the two tyrosines at the carboxy terminus of the α subunit were deleted. Fig. 2 A shows I-V curves of the NaIxK(ouab) difference currents for the ΔYY-truncated Na+/K+-ATPase α2 subunit. In contrast to ATP1A2 WT, the ΔYY construct mediated inwardly rectifying difference currents in the absence of extracellular K+. The amplitudes of these inward currents became smaller with increasing [K+]ext but were still observed for [K+]ext up to 2 mM at extremely negative potentials (−140 mV). At 10 mM K+, the ouabain-sensitive pump currents of the ΔYY construct were positive and increased steadily with voltage (Fig. 2 A). We then determined the full K+-induced NaIxK difference currents of the ΔYY construct at [Na+]ext = 100 mM by calculating the difference between currents recorded in the presence of a certain [K+]ext and those measured at [K+]ext = 0 (Fig. 2 B). The same information can be obtained by subtracting the NaI0K(ouab) currents at 0 [K+]ext from the NaIxK(ouab) currents (Fig. S1 A). Because in the case of the ΔYY construct extracellular K+ not only induces normal Na+/K+ outward pumping (as evident from the similarity to WT pump currents at positive potentials), but also diminishes the hyperpolarization-activated inward currents, the full K+-induced NaIxK currents (Fig. 2 B) were markedly different from those of the WT Na+/K+-ATPase (Fig. 2 D). For the WT enzyme, currents at [K+]ext = 10 mM increased steadily with voltage, and at [K+]ext below 5 mM, bell-shaped I-V curves were obtained with a maximum at +40 mV (Fig. 2 D). In contrast, the NaIxK currents of the ΔYY construct at 10 and 5 mM [K+]ext had a local minimum around −40 mV, and at lower [K+]ext, the I-V curves were maximal around −100 mV and decreased steadily between −100 and +60 mV (Fig. 2 B). Fitting of the [K+]ext-induced NaIxK currents at each membrane potential with a Hill equation resulted in NaK0.5(K+ext) values for the ΔYY construct (Fig. 2 C) and ATP1A2 WT (Fig. 2 E), which represent the voltage-dependent apparent affinities for extracellular K+ in the presence of 100 mM Na+ext. Characteristically U-shaped curves were obtained for both enzymes with similar minima, but the curve of the ΔYY construct was shifted by about −80 mV.


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)

Voltage and [K+]ext dependence of stationary currents at [Na+]ext = 100 mM. (A) I-V curves of normalized ouabain-sensitive K+-dependent currents NaIxK(ouab) of the Na+/K+-ATPase ΔYY deletion construct measured at 100 mM [Na+]ext and [K+]ext as indicated. (B) I-V curves of normalized K+-induced difference currents NaIxK (at 100 mM [Na+]ext) of the ΔYY construct. (C) Voltage-dependent NaK0.5(K+ext) values of the ΔYY construct from fits of a Hill function to the NaIxK currents in B at each membrane potential. The minimal NaK0.5(K+ext) was 1.02 ± 0.06 mM at −80 mV. The dashed line delineates the corresponding WT data from E for comparison. (D) I-V curves of normalized NaIxK difference currents for ATP1A2 WT with [K+]ext as indicated. (E) Voltage-dependent NaK0.5(K+ext) values for ATP1A2 WT from fits of a Hill function to the NaIxK currents in D at each membrane potential. The minimal NaK0.5(K+ext) was 1.10 ± 0.05 mM at 0 mV. Data for both WT and ΔYY are means ± SE from 14 cells of three oocyte batches. (F) I-V curves of normalized NaI10K(ouab) and NaI0K(ouab) currents of ATP1A2 WT and the ΔYY construct at pHext 7.4 ([Na+]ext = 100 mM). Data are means ± SE from six (WT) and seven (ΔYY) cells from two batches.
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fig2: Voltage and [K+]ext dependence of stationary currents at [Na+]ext = 100 mM. (A) I-V curves of normalized ouabain-sensitive K+-dependent currents NaIxK(ouab) of the Na+/K+-ATPase ΔYY deletion construct measured at 100 mM [Na+]ext and [K+]ext as indicated. (B) I-V curves of normalized K+-induced difference currents NaIxK (at 100 mM [Na+]ext) of the ΔYY construct. (C) Voltage-dependent NaK0.5(K+ext) values of the ΔYY construct from fits of a Hill function to the NaIxK currents in B at each membrane potential. The minimal NaK0.5(K+ext) was 1.02 ± 0.06 mM at −80 mV. The dashed line delineates the corresponding WT data from E for comparison. (D) I-V curves of normalized NaIxK difference currents for ATP1A2 WT with [K+]ext as indicated. (E) Voltage-dependent NaK0.5(K+ext) values for ATP1A2 WT from fits of a Hill function to the NaIxK currents in D at each membrane potential. The minimal NaK0.5(K+ext) was 1.10 ± 0.05 mM at 0 mV. Data for both WT and ΔYY are means ± SE from 14 cells of three oocyte batches. (F) I-V curves of normalized NaI10K(ouab) and NaI0K(ouab) currents of ATP1A2 WT and the ΔYY construct at pHext 7.4 ([Na+]ext = 100 mM). Data are means ± SE from six (WT) and seven (ΔYY) cells from two batches.
Mentions: Upon expression in Xenopus laevis oocytes, TEVC experiments were performed in Na+-containing buffers ([Na+]ext = 100 mM) to measure the K+ext-induced ouabain-sensitive pump currents of wild-type (WT) human α2/β1 Na+/K+-ATPase and of the ΔYY construct, in which the two tyrosines at the carboxy terminus of the α subunit were deleted. Fig. 2 A shows I-V curves of the NaIxK(ouab) difference currents for the ΔYY-truncated Na+/K+-ATPase α2 subunit. In contrast to ATP1A2 WT, the ΔYY construct mediated inwardly rectifying difference currents in the absence of extracellular K+. The amplitudes of these inward currents became smaller with increasing [K+]ext but were still observed for [K+]ext up to 2 mM at extremely negative potentials (−140 mV). At 10 mM K+, the ouabain-sensitive pump currents of the ΔYY construct were positive and increased steadily with voltage (Fig. 2 A). We then determined the full K+-induced NaIxK difference currents of the ΔYY construct at [Na+]ext = 100 mM by calculating the difference between currents recorded in the presence of a certain [K+]ext and those measured at [K+]ext = 0 (Fig. 2 B). The same information can be obtained by subtracting the NaI0K(ouab) currents at 0 [K+]ext from the NaIxK(ouab) currents (Fig. S1 A). Because in the case of the ΔYY construct extracellular K+ not only induces normal Na+/K+ outward pumping (as evident from the similarity to WT pump currents at positive potentials), but also diminishes the hyperpolarization-activated inward currents, the full K+-induced NaIxK currents (Fig. 2 B) were markedly different from those of the WT Na+/K+-ATPase (Fig. 2 D). For the WT enzyme, currents at [K+]ext = 10 mM increased steadily with voltage, and at [K+]ext below 5 mM, bell-shaped I-V curves were obtained with a maximum at +40 mV (Fig. 2 D). In contrast, the NaIxK currents of the ΔYY construct at 10 and 5 mM [K+]ext had a local minimum around −40 mV, and at lower [K+]ext, the I-V curves were maximal around −100 mV and decreased steadily between −100 and +60 mV (Fig. 2 B). Fitting of the [K+]ext-induced NaIxK currents at each membrane potential with a Hill equation resulted in NaK0.5(K+ext) values for the ΔYY construct (Fig. 2 C) and ATP1A2 WT (Fig. 2 E), which represent the voltage-dependent apparent affinities for extracellular K+ in the presence of 100 mM Na+ext. Characteristically U-shaped curves were obtained for both enzymes with similar minima, but the curve of the ΔYY construct was shifted by about −80 mV.

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