Meier S et al. (2010), Hyperpolarization-activated inward leakage curr...

XB-ART-41054

J Gen Physiol 2010 Feb 01;1352:115-34. doi: 10.1085/jgp.200910301.

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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 .

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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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Species referenced: Xenopus
Genes referenced: atp1a2 tbx2

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	Figure 1. 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.
	Figure 2. 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.
	Figure 3. Voltage and [K+]ext dependence of stationary currents in the absence of Na+ext. (A and B) I-V curves of normalized K+-dependent NMDGIxK difference currents of the ΔYY construct (A) and ATP1A2 WT (B) in the absence of extracellular Na+ ([NMDG+]ext = 100 mM, pH 7.4) with [K+]ext as indicated. Also shown in A and B are the ouabain-sensitive currents (NMDGIouab) in the absence of K+ for the ΔYY construct and ATP1A2 WT. (C) K0.5(K+ext) values for the ΔYY construct and for ATP1A2 WT at [Na+]ext = 0. For each construct, data are means ± SD from eight oocytes out of two batches.
	Figure 4. Properties of transient currents and hyperpolarization-induced inward currents of the ΔYY construct. (A and B) Ouabain-sensitive presteady-state currents NaI(ouab) at [Na+]ext = 100 mM and [K+]ext = 0, pHext 7.4, upon voltage steps from −30 mV to potentials between +60 and −140 mV (in 20-mV decrements) for WT (A) and the ΔYY construct (B). (C and D) Voltage dependence of reciprocal time constants from ON transient currents for ATP1A2 WT (C) and ΔYY (D). Data for WT and ΔYY are means ± SE from 14 cells out of three batches. Results from a fit of a polynomial function to the WT or ΔYY data are superimposed in C and D as a dashed or dotted line, respectively. (E) Q-V curves obtained from QOFF charges of transient currents from 14 cells expressing ATP1A2 WT. The corresponding QON charges were equivalent (not depicted). The fit of a Boltzmann function to Q-V distribution is superimposed as a dashed line (fit parameters: Qmin = −6.65 ± 0.12 nC; Qmax = 2.34 ± 0.04 nC; V0.5 = −0.5 ± 1.0 mV; zq = 0.76 ± 0.02). (F) Q-V curves obtained from QON and QOFF charges of 14 cells expressing the ΔYY construct. The fit of a Boltzmann function to the QOFF values is superimposed as a dashed line (fit parameters: Qmin = −1.14 ± 0.17 nC; Qmax = 4.72 ± 0.63 nC; V0.5 = −83.4 ± 7.9 mV; zq = 0.81 ± 0.06). The short dashed line represents the inverted QOFF fit curve for comparison with QON values. QON and QOFF values of the ΔYY construct were significantly different at hyperpolarizing potentials (, P > 0.05; *, P > 0.005; Student’s t test). The WT Q-V curve from E is included as a dotted line in F after appropriate scaling. (G) Transient currents in response to ON voltage steps to +60 and −140 mV from WT data in A and ΔYY data in B on an expanded time scale. The voltage step occurred at 58 ms (dashed line). Fits of the sum of two exponential functions to the data (from t0 = 59 ms) yielded time constants for the fast rise, τ1, and the slower decay, τ2. The peak of the transient current of ΔYY at −140 mV appeared with a delay of ∼2 ms (gray bars). (H) [Na+]ext dependence of normalized ouabain-sensitive inward currents of the ΔYY construct at [K+]ext = 0. The shallow dashed line shows the I-V curve of NaI(ouab) currents of ATP1A2 WT at [Na+]ext = 100 mM and [K+]ext = 0. For each construct, data are means ± SE from 14 cells of four batches. (I) [Na+]int dependence of ouabain-sensitive NaI(ouab) currents (at [Na+]ext = 100 mM and [K+]ext= 0), measured on an oocyte coexpressing the ΔYY construct and the amiloride-sensitive Na+ channel ENaC. Na+ loading was achieved by exposing oocytes to [Na+]ext-containing solutions in the absence of amiloride, and [Na+]int was determined after each loading step from the amiloride-sensitive reversal potential shift (see Materials and methods). Data from one out of three experiments with a similar [Na+]int range are shown. (J) Temperature dependence (Arrhenius plots) of Na+/K+ pump currents (NaI10K) at 0 mV (filled squares) and hyperpolarization-induced inward currents (NaI(ouab)) at −140 mV (open squares) of the ΔYY construct. Data are from one out of four experiments across a similar temperature range (18–30°C). For both data sets, the amplitudes were normalized to the respective current at 21°C. Activation energies were derived from linear fits to the data.
	Figure 5. Voltage dependence of stationary and ouabain-sensitive transient currents of the ΔYY construct under different ionic conditions and pHext. (A and B) Current recordings at −30 mV from a ΔYY-expressing oocyte in the presence (A) or absence (B) of extracellular Na+ at pH 7.4 and 5.5. If present, [Na+]ext and [NMDG+]ext were 100 mM, and [K+]ext and [ouabain] were 10 mM. The composition of the extracellular solution is indicated above the current traces. Dashed lines indicate the “zero pump current” level (at 10 mM ouabain) as a reference for pump-related currents. (C and D) I-V curves of stationary currents in different extracellular solutions from oocytes expressing ΔYY (C) or ATP1A2 WT (D), each normalized to the NaI10K(ouab) current at 0 mV. Similar data were obtained on at least three different cells. (E) Ouabain-sensitive transient currents of ΔYY at pH 7.4 (NMDGI(ouab) and NaI(ouab)) and pH 5.5 (NMDGI(ouab)5.5 and NaI(ouab)5.5) in the absence of K+ext upon 250-ms voltage steps from −30 to −140 mV. The peak of the NaI(ouab) current is indicated by a dotted line. Note the time axis break (hatched bar).
	Figure 6. Properties of stationary currents of ATP1A2 mutants YY-FF and YY-AA. (A and B) Normalized I-V curves for NaIxK currents of ATP1A2 mutants YY-FF (A) and YY-AA (B), with [K+]ext as indicated ([Na+]ext = 100 mM, pH 7.4). (C and D) NaK0.5(K+ext) values from NaIxK currents for mutant YY-FF (C) and YY-AA (D) from fits of a Hill function to the data in A and B at each membrane potential. The corresponding WT data from Fig. 2 E (dashed lines in C and D) and ΔYY data from Fig. 2 C (dotted lines in C and D) are superimposed. (E) I-V curves of normalized ouabain-sensitive NaI(ouab) currents ([Na+]ext = 100 mM and [K+]ext = 0, pH 7.4) for ATP1A2 WT and mutants YY-FF, YY-AA, and ΔYY. Data are means ± SE of 10–14 cells from three batches.
	Figure 7. Properties of transient currents for mutants YY-FF and YY-AA. (A and B) Voltage dependence of reciprocal time constants from ON transient currents for mutant YY-FF (A) and YY-AA (B) ([Na+]ext = 100 mM and [K+]ext = 0, pH 7.4). Data are means ± SE from 13 (YY-FF) or 10 (YY-AA) oocytes from at least three batches. Curves delineating the corresponding WT data from Fig. 4 C (dashed lines in A and B) and the ΔYY data from Fig. 4 D (dotted line in B) are superimposed. (C and D) Q-V curves for QOFF charge integrals from oocytes expressing ATP1A2 mutants YY-FF (C) and YY-AA (D). Dashed lines show fits of a Boltzmann function with the following parameters: YY-FF (C): Qmin = −2.73 ± 0.05 nC; Qmax = 2.26 ± 0.03 nC; V0.5 = −27.1 ± 0.8 mV; zq = 0.81 ± 0.02; YY-AA (D): Qmin = −1.15 ± 0.13 nC; Qmax = 5.59 ± 0.15 nC; V0.5 = −120 ± 18 mV; zq = 0.44 ± 0.03. The Q-V distribution of ATP1A2 WT from Fig. 4 E is superimposed as a dotted line in C and D after appropriate scaling.
	Figure 8. Partial reaction schemes for the Na+ transport limb of the Na+/K+-ATPase cycle. Scheme 1 was introduced by Holmgren and Rakowski (2006) to describe the dependence of Na+/K+-ATPase presteady-state currents on intracellular [Na+]. At saturating intracellular concentrations of Na+ and ATP, the intracellular Na+ binding (and phosphorylation) is at rapid equilibrium, and the subsequent slow occlusion step kinetically isolates the intracellular Na+ binding step from the more rapid extracellular Na+ release steps. Scheme 2 refers to Vasilyev et al. (2004), who provided a concept for the mainly H+-driven inward currents of the Na+/K+-ATPase observed in Na+- and K+-free extracellular solutions. At low pHext, the energy barrier for equilibration of occluded Na+ ions with the intracellular space is reduced so that the occluded state can be reached from the extracellular as well as the intracellular solution. See Appendix for details.

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