Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism

The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCP^O to the red OCP^R form. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP’s N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

     

Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2

Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic hemiplegic migraine, including missense mutations (T263M, T376M, R383H, A606T, R763H, M829R, R834Q, R937P, and X1021R), a deletion mutant (del(K935-S940)ins(I)), and a frameshift mutation (S966fs). According to the Na(+)/K(+)-ATPase crystal structure, a subset of the mutated residues (Ala(606), Arg(763), Met(829), and Arg(834)) is involved in important interdomain H-bond networks, and the C terminus of the enzyme, which is elongated by the X1021R mutation, has been implicated in voltage dependence and formation of a third Na(+)-binding site. Upon heterologous expression in Xenopus oocytes, the analysis of electrogenic transport properties, Rb(+) uptake, and protein expression revealed pronounced and markedly diverse functional alterations in all ATP1A2 mutants. Abnormalities included a complete loss of function (T376M), impaired plasma membrane expression (del(K935-S940)ins(I) and S966fs), and altered apparent affinities for extracellular cations or reduced enzyme turnover (R383H, A606T, R763H, R834Q, and X1021R). In addition, changes in the voltage dependence of pump currents and the increased rate constants of the voltage jump-induced redistribution between E(1)P and E(2)P states were observed. Thus, mutations that disrupt distinct interdomain H-bond patterns can cause abnormal conformational flexibility and exert long range consequences on apparent cation affinities or voltage dependence. Of interest, the X1021R mutation severely impaired voltage dependence and kinetics of Na(+)-translocating partial reactions, corroborating the critical role of the C terminus of Na(+)/K(+)-ATPase in these processes.     

Control of gastric H,K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E(1)P and E(2)P states and measured Rb(+) uptake under various ionic and pH conditions. The steady-state E(1)P/E(2)P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb(+) uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E(1)P/E(2)P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V(0.5), the voltage, at which the E(1)P/E(2)P ratio is 50∶50, by -100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E(1)P→E(2)P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb(+) uptake yielded an activation energy of ～90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E(1)P→E(2)P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na(+) profoundly alters the voltage-dependent E(1)P/E(2)P distribution indicating that Na(+) ions can act as surrogates for protons regarding the E(2)P→E(1)P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ～0.5 and ～0.2, respectively.     

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

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 α₂ subunit decreases the affinity for extracellular and intracellular Na⁺, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na⁺ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K⁺, the ΔYY 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 ΔYY 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 α subunit represents a structural and functional relay between Na⁺ binding site III and the intracellular cation occlusion gate.     

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCP^O) into the red-shifted, active state (OCP^R) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCP^R is converted to OCP^O and dissociates from PBs; however, the mode and site of OCP^R/FRP interactions remain elusive. Recently, we have introduced the purple OCP^W288A mutant as a competent model for the signaling state OCP^R (Sluchanko et al., Biochim Biophys Acta 1858:1–11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCP^W288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCP^O, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCP^O, a substantial compaction of the OCP^W288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCP^W288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.     

E2P State Stabilization by the N-terminal Tail of the H,K-ATPase ��-Subunit Is Critical for Efficient Proton Pumping under in Vivo Conditions

The catalytic α-subunits of Na,K- and H,K-ATPase require an accessory β-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the β-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated β-variants were expressed in Xenopus oocytes, together with the S806C α-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E₁P/E₂P states. Whereas the E₁P/E₂P conformational equilibrium was unaffected for the shorter N-terminal deletions βΔ4 and βΔ8, we observed significant shifts toward E₁P for the two larger deletions βΔ13 and βΔ29. Moreover, the reduced ΔF/F ratios of βΔ13 and βΔ29 indicated an increased reverse reaction via E₂P → E₁P + ADP → E₁ + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E₂P-specific inhibitor {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080, which becomes especially apparent at high concentrations (100 μm). Despite unaltered apparent Rb⁺ affinities, the maximal Rb⁺ uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the β-N terminus and α-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase β-subunit may stabilize the pump in the E₂P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the β-N terminus of the closely related Na,K-ATPase does not affect the E₁P/E₂P distribution or pump activity, indicating that the E₂P-stabilizing effect by the β-N terminus is apparently a unique property of the H,K-ATPase.The gastric H,K-ATPase fulfills the remarkable task of pumping protons against a more than 10⁶-fold concentration gradient. H⁺ extrusion is coupled to countertransport of an equal number of K⁺ ions for each ATP molecule hydrolyzed, resulting in an electroneutral overall process (1). Characteristic for all P-type ATPases, the enzyme cycles between the two principal conformational states (E₁ and E₂) and the corresponding phosphointermediates (E₁P and E₂P), which are formed by reversible phosphorylation of an aspartate residue in the highly conserved DKTGTLT motif. According to a Post-Albers-like reaction scheme (see Fig. 1A), the conformational E₁P → E₂P transition converts the high H⁺/low K⁺ affinity of the cation binding pocket into a low H⁺/high K⁺ affinity binding site, hence enabling proton release into the stomach lumen and subsequent binding of extracellular K⁺. Because the pump faces a lumenal proton concentration of ∼150 mm (2), proton release is probably the energetically most demanding step in the reaction cycle. Thus, during the conformational E₁P → E₂P transition, enormous pK_a changes of the H⁺-coordinating residues have to occur that most likely involve the rearrangement of a positively charged lysine side chain (Lys-791 in rat H,K-ATPase) (3).Open in a separate windowFIGURE 1.Post-Albers scheme (A) and cryo-EM structural representation of pig gastric H,K-ATPase in the fluoroaluminate-bound pseudo-E₂P state (B). A, Post-Albers scheme of the proposed reaction cycle of the gastric H,K-ATPase. E₁P/E₂P conformational states giving rise to voltage jump-induced fluorescence changes of TMRM-labeled H,K-ATPase molecules are highlighted (gray box). B, structural representation based on the cryo-EM structure of the pig gastric H,K-ATPase (surface or mesh, contoured at 1 σ; EM Data Bank code 5104) and the corresponding homology model (schematic; Protein Data Bank code 3IXZ). Inset, a close-up view (from the right side of the molecule) showing the putative interaction sites of the β-subunit N terminus with the P-domain (red arrow) and αTM3 (black arrow), respectively. Color coding is indicated in the figure.All P₂-type ATPases share a common catalytic α-subunit, composed of 10 transmembrane domains harboring the ion-binding sites and a large cytoplasmic loop with the nucleotide-binding domain, the phosphorylation domain (P-domain),² and the actuator domain (A-domain) (4). However, a unique feature of K⁺-transporting Na,K- and H,K-ATPase enzymes is the requirement for an accessory β-subunit, which is indispensable for proper folding, maturation, and plasma membrane delivery (5, 6). Despite only 20–30% overall sequence identity between the H,K-ATPase β-subunit and the Na,K β-isoforms, the topogenic structure is similar: a short N-terminal cytoplasmic tail, followed by a single transmembrane segment and a large extracellular C-terminal domain with glycosylation sites and disulfide-bridging cysteines. Numerous studies have demonstrated that the β-subunit of the Na,K-ATPase is more than just a chaperone for the α-subunit, being also required for proper ion transport activity of the holoenzyme. In fact, it has been discovered that different cell- and tissue-specific β-isoforms have distinct effects on the cation affinities (7–9). Furthermore, it was shown that mutational changes in all three topogenic domains of the Na,K-ATPase β-subunit (10–19) as well as chemical interference with disulfide-forming cysteines in the Na,K-ATPase β-subunit ectodomain (20–22) affect the cation transport properties of the sodium pump. Finally, conformational changes in the β-subunit during the Na,K-ATPase reaction cycle were demonstrated by proteolytic digestion studies (23) and voltage clamp fluorometry (24).Far less is known about the functional significance of the single H,K-ATPase β-isoform, especially about its potential impact on cation transport (reviewed in Refs. 25 and 26). We have proven recently that E₂P state-specific transmembrane interactions between residues in αTM7 and two highly conserved tyrosines in the βTM of both Na,K- and H,K-ATPase significantly stabilize the E₂P conformation (19). Mutational disruptions of this interaction resulted in substantial shifts toward E₁P and severely affected H⁺ secretion, which highlighted the physiological relevance of this E₂P state stabilization. Notably, according to the recently published cryo-EM structure of pig gastric H,K-ATPase in the pseudo-E₂P state (27), the N-terminal tail of the β-subunit makes direct contact with the phosphorylation domain of the α-subunit (see Fig. 1B), thus indicating an additional E₂P state stabilization mediated by the β-N terminus. Although this idea was further supported by biochemical studies on N-terminally truncated mutants, direct evidence for this putative E₂P-stabilizing interaction and its potential significance for ion transport in intact cells is still lacking.Here, we demonstrate for the first time the functional importance of the gastric H,K-ATPase β-subunit N terminus in living cells under in vivo conditions: voltage clamp fluorometry, Rb⁺ flux, and {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 sensitivity measurements revealed E₁P-shifted, ion transport-impaired phenotypes for two N-terminally truncated H,K β-variants, thus substantiating the E₂P-stabilizing effect of the β-N terminus suggested by the recent cryo-EM structure.     

Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes

The role of N-linked glycosylation of beta-subunits in the functional properties of the oligomeric P-type ATPases Na,K- and H,K-ATPase has been examined by expressing glycosylation-deficient Asn-to-Gln beta-variants in Xenopus oocytes. For both ATPases, the absence of the huge N-linked oligosaccharide moiety on the beta-subunit does not affect alpha/beta coassembly, plasma membrane delivery or functional activity of the holoenzyme. Whereas this is in line with several previous glycosylation studies on Na,K-ATPase, this is the first report showing that the cell surface delivery and enzymatic activity of the gastric H,K-ATPase is unaffected by the lack of N-linked glycosylation. Sulfhydryl-specific labeling of introduced cysteine reporter sites with the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes enabled us to further investigate potential effects of the N-glycans on more subtle enzymatic properties, like the distribution between E 1P/E 2P states of the catalytic cycle and the kinetics of the E 1P/E 2P conformational transition under presteady state conditions. For both Na,K-ATPase and H,K-ATPase, we observed differences in neither the voltage-dependent E 1P/E 2P ratio nor the kinetics of the E 1P/E 2P transition between holoenzymes comprising glycosylated and glycosylation-deficient beta-subunits. We conclude that the N-linked glycans on these essential accessory subunits of oligomeric P-type ATPases are dispensable for proper folding, membrane stabilization of the alpha-subunit and transport function itself. Glycosylation is rather important for other cellular functions not relevant in the oocyte expression system, such as intercellular interactions or basolateral versus apical targeting in polarized cells, as demonstrated in other expression systems.