Измерение Катионит транспорта по Na, K и H, K-АТФазы<em&gt; Xenopus</em&gt; Ооцитов методом атомно-абсорбционной спектрофотометрии: Альтернатива радиоизотопных Анализы

Резюме

We describe a method to quantify the activity of K⁺-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb⁺ or Li⁺ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Аннотация

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.

Введение

We wanted to develop a sensitive, safe and inexpensive alternative to radioactive tracer experiments to investigate the specific transport activity of ion translocating membrane proteins in order to circumvent restrictions regarding the access to isotope laboratories, safety requirements or the use of costly radioisotopes, which - as in the case of lithium - may even be unavailable due to extremely short decay times. We were particularly interested in determining the activity of the electroneutrally operating gastric H⁺,K⁺-ATPase, because the enzyme does not generate current and its activity can therefore not be addressed by electrophysiological methods. Since Na⁺,K⁺- and H⁺,K⁺-ATPase transport Rb⁺ as efficient as K⁺ (and Li⁺ as well), the high sensitivity of the AAS technique for rubidium or lithium should facilitate sensitive detection of transport activity. Atomic absorption spectrophotometers are common analytical devices, which are widely distributed in chemical laboratories and should be accessible to a large number of interested scientists. Furthermore, we wanted to take advantage of the Xenopus oocyte expression system, which utilizes large single cells (about 1.0-1.5 mm diameter) that allow to achieve a remarkably low cell-to-cell variability regarding the protein expression level within a single batch. A simple calculation demonstrates the feasibility of the AAS assay: The detection limit (characteristic mass) for rubidium with the THGA-AAS technique is 10 pg or 1.2·10^-13 mol (Rb: 85.47 g/mol), for lithium 5.5 pg or 7.9·10^-13 mol (Li: 6.94 g/mol). Upon heterologous expression of Na⁺,K⁺-ATPase in Xenopus oocytes, pump currents of 100 nA can be achieved (which equals about 6.2·1011 elementary charges per second, or 1.03·10^-12 mol s^-1), thus resulting in a transport of 6·10^-6 C of charge within 1 min. Since the transport of one net charge corresponds to the uptake of two Rb⁺ ions (due to the 3Na⁺/2K⁺ stoichiometry), 100 nA current for 1 min corresponds to an uptake of 1.2·10^-10 mol Rb⁺. Thus, even upon a 1,000-fold dilution (homogenization of an oocyte with about 1 μl volume in 1 ml water), a typical THGA-AAS sample (20 μl) contains 2.4·10^-12 mol Rb⁺ (or 204 pg), which is far above the detection threshold. Therefore, even transporters with more than 100-fold lower transport activity or plasma membrane expression can be assayed with the technique by appropriately adjusting the flux time of the experiment.

Since the pumping rate is sensitively dependent on temperature (typical activation energies for Na⁺,K⁺-ATPase are in the range of 90 kJ/mol to 130 kJ/mol^1-3, which results in an about 30% increase in the turnover rate upon a change from 20 °C to 22 °C), it is mandatory to carry out the flux measurements under precise temperature control (air conditioning) with well equilibrated buffer solutions. Furthermore, oocytes should be carefully selected regarding homogenous size for the expression of an ion transporter. With these precautions, it is possible to routinely achieve experimental standard errors of less than 10 percent with about 10 cells per experimental condition. Using this technique, we were able to determine e.g. the apparent Rb⁺ affinities of cation transport^4-6, the influence of extra- and intracellular pH⁷ and the effect of mutations of residues involved in cation coordination during transport^4,8. An advantage of the technique is that ion fluxes can also be determined in combination with two-electrode voltage clamping of the oocytes, which on one hand assures accurate control of the membrane potential during transport and on the other hand allows to correlate ion flux with membrane current. Thus, it was possible to verify the 3Na⁺/2K⁺ stoichiometry of the Na⁺,K⁺-ATPase (see exemplary results below) and to determine the voltage dependence of cation transport of the gastric H⁺/K⁺-ATPase⁷.

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протокол

1. cDNA Constructs and Protein Expression in Xenopus Oocytes

The cDNA of the membrane protein of interest should be cloned into a vector suitable for expression in Xenopus laevis oocytes such as pTLN⁹ or pcDNA3.1X¹⁰. Such optimized vectors contain the 5'- and 3'-untranslated regions (UTR) of the Xenopus laevis β-globin gene flanking the multiple cloning site (MCS), an RNA polymerase promoter sequence (pTLN: SP6, pcDNA3.1X: T7) located before the 5' UTR, a poly-A stretch in 3' direction of the MCS to ensure cRNA stability in cells, and further downstream another sequence of single-cutting restriction endonuclease sites for linearization of the plasmid in order to serve as a template for in vitro cRNA transcription.

To distinguish the activity of the overexpressed human Na⁺/K⁺-ATPase (α₂-subunit+β₁-subunit) from the endogenous Na⁺/K⁺-ATPase of the oocytes, the mutations Q116R and N127D were introduced to obtain an ouabain-resistant protein with an IC50 in the millimolar range¹¹. In the case of H⁺/K⁺-ATPase, flux measurements were carried out using the H⁺/K⁺-ATPase α-subunit mutant S806C, since Rb⁺ fluxes were correlated with kinetic data from voltage-clamp fluorometry (VCF) experiments. The VCF technique (https://www.jove.com/video/2627/ examining-the-conformational-dynamics-of-membrane-proteins-in-situ-with-site-directed-fluo rescence-labeling)¹² is based upon site-specific attachment of a fluorophore to a strategically introduced cysteine residue, which reports conformational changes of the enzyme.

2. Linearization and Purification of the DNA Template

1. For linearization of the DNA template, digest 3 μg of plasmid DNA in 50 μl reaction volume with 5 μl 10x-concentrated buffer appropriate for the restriction enzyme and 10 U of restriction enzyme for 1 hr at 37 °C.
2. Purify the linearized DNA using the High-Pure PCR Product Purification Kit (Roche Applied Science). First, add 350 μl binding buffer (containing guanidiniumisothiocyanate) to the digest vial. After this purification step, the linearized DNA template is 'RNA grade' because guanidiniumisothiocyanate is an efficient protein denaturant and destroys in particular RNAse, which is a frequent contaminant in molecular biological laboratories.
3. Insert spin columns into collection tubes, load the column with DNA solution from step 2, centrifuge at 13,000 x g for 30 sec and discard flow-through.
4. Add 500 μl of washing buffer to the column. Centrifuge 1 min at 13,000 x g and discard flow through.
5. Add 200 μl of washing buffer to the column. Centrifuge 1 min at 13,000 x g to fully dry the column.
6. Place the spin column in a fresh 1.5 ml Eppendorf tube and add 50 μl of nuclease-free water (Ambion Product # AM9937) to the column. Elute by centrifuging for 1 min at maximum speed.
7. After elution, concentrate the DNA-containing solution to about 15 μl. This step ensures that the solution contains 0.5 μg linearized DNA in 3 μl, which is the recommended concentration for the following in vitro cRNA transcription reaction.

3. In Vitro cRNA Synthesis

1. Choose the appropriate mMessage mMachine Kit (Ambion Product #1340, #1344) according to the promoter sequence on the plasmid (SP6, T7). In 10 μl total reaction volume, add 3 μl linearized DNA from the previous step and 1 μl transcription buffer to pre-aliquoted 5 μl-samples of NTP-cap mix. Pre-aliquoting of the NTP-cap mix is necessary to avoid frequent freeze-thaw-cycles, which may lead to degradation of the nucleoside-triphosphates. Finally, add 1 μl of the appropriate RNA polymerase, mix briefly and spin-down the reaction mix.
2. Incubate the reaction mix for 1 to 2 hr at 37 °C. The incubation time should not exceed 2 hr, because this may lead to lower cRNA yield or quality.
3. Add 15 μl of ice-cold 3 M LiCl solution from the kit, 15 μl cold nuclease-free water, mix briefly (do not vortex), briefly spin down the sample at 11,000 x g for 10 sec, and store at -20 °C for at least 30 min to allow precipitation of the cRNA.
4. Pre-cool centrifuge to 4 °C and centrifuge at least 15 min at 13,000 x g after precipitation
5. Remove the supernatant carefully and completely from the pale brown pellet at the bottom of the tube and add 150 μl of 70% RNA-grade ethanol cooled to -20 °C. Centrifuge at 4 °C for 5 min at 13,000 x g.
6. Remove the supernatant completely, briefly dry (1-2 min) the remaining cRNA pellet in a centrifugal evaporator (e.g. Eppendorf Concentrator model 5301) at room temperature.
7. Dissolve the pellet in 12 μl of cold nuclease-free water. CAUTION: All subsequent steps should be carried out with the cRNA sample stored on ice.
8. Determine the cRNA concentration using a Spectrophotometer (e.g. Eppendorf BioPhotometer).
9. Check the integrity of the cRNA by agarose gel electrophoresis. CAUTION: This cross-check is necessary, since even partially degraded RNA yields non-zero UV absorption. For cRNA electrophoresis, use a thoroughly cleaned electrophoresis chamber newly filled with TAE buffer and a freshly prepared 1% agarose gel. If RNAse contamination is suspected in the electrophoresis chamber or buffer, fill the chamber with 0.1 M HCl for 1 hr for cleaning and prepare TAE buffer with nuclease-free water.
10. Store cRNA at -20 °C until use. cRNA can be stored for at least one year at -20 °C.

4. Obtaining Ovary Material from Xenopus laevis Females by Partial Ovariectomy

This protocol has been approved by the responsible state authority (Landesamt für Gesundheit und Soziales Berlin, The Senate of Berlin, Germany).

1. Do not feed the frog within at least 12 hr prior to surgery to prevent vomiting.
2. Prepare a solution of 2 g of the local anesthetic tricaine (MS-222) dissolved in 1 L water adjusted to pH 7.0 with 5 mM HEPES buffer. CAUTION: Wear nitrile gloves at all times while handling MS-222 and prepare the solution in a chemical safety cabinet.
3. Immerse the frog in the tricaine solution (max. 10 min) until the animal no longer shows righting reflexes. To check unresponsiveness, try to turn the frog on its back in your hands and watch for defense movements.
4. Take the frog out of the tricaine solution by hand and rinse the animal with cold tap water to remove the anesthetic.
5. Place the frog with its back on top of a bed of ice covered with a wet paper towel to avoid damage to the frog's skin by direct contact to ice. Keep the skin of the animal wet at all times during the operation to avoid drying. Placement on ice prolongs the time of anesthesia.
6. Make a small abdominal incision (8-10 mm length) with sharp scissors on either the left or the right side of the frog's midline. CAUTION: Be careful not to injure the lateral line. Subsequent surgeries should be made on alternating sides to allow for good recovery of the underlying ovary tissue. Usually, three layers of tissue (the outer skin, a thin layer of white connective tissue - the fascia - and a muscle layer) have to be penetrated.
7. After opening the abdomen, seize parts of ovarian lobes with blunt forceps and pull gently to the exterior. As a guideline, remove about 3-5 cm of ovary material from the ovary by incision. In case of injury of small blood vessels and bleeding, apply pressure with a sterile plastic rod or cool the injury with ice until the bleeding stops.
8. Surgically close the incision using an interrupted suture pattern with 4.0 Ethicon Vicryl suture (Johnson & Johnson #V633H). Use surgical instruments for suturing and tie two or three knots separately along the incision. CAUTION: It is important for each knot to suture the three tissue layers in one step.
9. After operation, place the frog into a slightly inclined water basin (30 L), which contains just enough water to cover its bottom so that the animal's nostrils are above water level to permit breathing. Cover the animal during the wake-up period (about 1-2 hr) with a wet paper towel to avoid drying of the sensitive skin.
10. When the animal is active again, fill the water basin and keep the frog separately from the other animals for 3-4 days to monitor recovery and wound healing.
11. After surgery, save the frog from subsequent operations for at least 3 months to allow for complete recovery of the animal and its ovarian tissue. As a guideline, a maximum of 6-8 surgeries can be performed on each frog.

5. Enzymatic Isolation of Individual Oocytes

1. Cut the ovarian material into smaller pieces with scissors and transfer the tissue into Oocyte Ringer Solution without Ca²⁺ (110 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM HEPES, pH 7.4) containing collagenase (20 mg/ml, type 1A from Clostridium hystolyticum, Sigma #C9891) and trypsin inhibitor (10 mg/ml, Type III-O from chicken egg white, Sigma #T2011).
2. Gently shake the digest solution for 2.5 hr at 18 °C. Afterwards, follow the progress of the digest in 15 min intervals until most oocytes are separated from the follicular tissue.
3. Wash the oocytes several times in a 50 ml Falcon tube with Oocyte Ringer Solution without Ca²⁺ until the solution is sufficiently cleared from tissue debris.
4. Store the oocytes in Oocyte Ringer Solution containing Ca²⁺ (110 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM HEPES, pH 7.4) including gentamycin (50 μg/ml) at 18 °C.

6. cRNA Injection

1. For expression of Na⁺,K⁺- or H⁺,K⁺-ATPase, inject oocytes with 50 nl cRNA solution containing 25 ng α-subunit and 5 ng β-subunit cRNA using an electrically driven Nanoject II injection pump (Drummond Scientific Inc.), with 3.5" hematocrit tubes (Drummond #3 000-203-G/X). CAUTION: The amount of mRNA may vary according to the target protein and might need to be adjusted for maximal expression by Western blotting.
2. For each flux condition (e.g. at a specific Rb⁺ concentration), inject 18-22 oocytes. From these, 50% will be assayed under "+flux" conditions (e.g. a certain Rb⁺ concentration), and 50% under "-flux" conditions (e.g. with the chosen Rb⁺ concentration and a specific inhibitor of the enzyme in saturating concentration, such as 10 mM ouabain for Na⁺,K⁺-ATPase or 10 μM SCH28080 at pH 5.5 for H⁺,K⁺-ATPase).
3. After injection, transfer oocytes separately into 96-well plates half-filled with Oocyte Ringer Solution containing Ca²⁺ and 50 μg/ml gentamycin at 18 °C.
4. Cover the 96-well plate with Parafilm to prevent excessive evaporation.
5. Incubate the oocytes for 2-7 days (depending on the protein of interest) at 18 °C. If oocyte storage for more than 3 days is required, it is recommended to exchange the incubation buffer within the microwells every 3 days.
6. For each set of flux data (e.g. a [Rb⁺] titration experiment), about 10 non-injected (or injected with 50 nl nuclease-free water) control oocytes should be assayed under "+flux" and "-flux" conditions. Thus, incubate a sufficient number of control cells in the same way as cRNA-injected oocytes prior to flux analysis.

Pretreatment of heterologous ATPase-expressing oocytes

Na⁺ loading procedure

For functional measurements of Na⁺/K⁺-ATPase, it is important to elevate the intracellular Na⁺ concentration¹³, as follows:

1. Transfer oocytes into Na⁺ Loading Buffer (110 mM NaCl, 2.5 mM Na-citrate, 2.5 mM MOPS, 2.5 mM TRIS, pH 7.4) for 45 min on ice.
2. For subsequent recovery, incubate oocytes in Post Loading Buffer "PLB" (100 mM NaCl, 1 mM CaCl₂, 5 mM BaCl₂, 5 mM NiCl₂ and 2.5 mM MOPS, 2.5 mM TRIS, pH 7.4) for at least 30 min on ice.
3. Store oocytes in PLB on ice until use.

7. Pre-blocking of Endogenous Na⁺,K⁺-ATPase

CAUTION: Since Xenopus oocytes express an endogenous Na⁺,K⁺-ATPase, it is important to block this endogenous pump prior to Rb⁺ flux experiments, as follows:

1. Now to block the endogenous Na⁺,K⁺-ATPase by incubating the oocytes in Post Loading Buffer containing 100 μM ouabain for 15 min at room temperature.

8. Electrophysiology

1. Prepare microelectrodes by pulling borosilicate capillaries (GB150F-8P, Science Products GmbH) using a two-step micropipette puller (Model PC-10, Narishige).
2. Fill glass electrodes from the back with 3 M KCl. For TEVC experiments with H⁺,K⁺-ATPase, we recommend to use 3M NaCl instead, which led to more stable current recordings. Mount pipette over the Ag/AgCl wire of the microelectrode holders of the two-electrode voltage-clamp setup.
3. Insert the glass microelectrodes and two agar bridges (these should be made with NaCl/agar instead of commonly used KCl/agar), which connect the Ag/AgCl reference electrodes to the bath solution into the recording chamber (Warner Instr., model RC-10) pre-filled with measuring buffer (e.g. PLB with 10 μM ouabain for Na⁺,K⁺-ATPase).
4. Switch the voltage-clamp amplifier (Turbo TEC-10CX, NPI Electronics, Tamm, Germany) into the "OFF" mode.
5. Check the resistance of each glass electrode separately (both should be in the range of 0.5-2 MΩ).
6. Check offset potentials of voltage and current electrode on the appropriate TEVC amplifier display. In case of discrepancy from zero adjust the offset with the corresponding offset regulators of the amplifier.
7. Place an oocyte in the middle of the chamber and penetrate the membrane gently with the current and the voltage electrode.
8. Check potential reading of the amplifier. The reading for the potential of the voltage electrode now gives the value for the resting potential of the cell.
9. Switch amplifier to VC (voltage-clamp) mode.
10. Using the pCLAMP software (Molecular Devices Corp., Sunnyvale, CA), clamp the cell to a pre-defined membrane potential. This can be either the value of the previously measured resting potential (for recording under zero membrane current conditions) or any desired test potential value. Pay attention to the amplitude and stability of the leak current (in the absence of K⁺/Rb⁺/Li⁺), which - as a general guideline - should not exceed -200 nA at -30 mV.
11. Perfuse the oocyte for a certain time (e.g. 3 min for Na⁺,K⁺-ATPase) with Rb⁺-containing solution (to achieve maximal current use saturating concentrations from flux measurements under non-clamped conditions) and record pump current with pCLAMP software in the continuous recording mode. Make sure that Rb⁺ uptake is completely stopped afterwards by perfusing the cell with Rb⁺-free solution for 30 sec.
    1. Stop the two-electrode voltage-clamp experiment, remove the oocyte from the recording chamber and proceed to the washing step 4 of paragraph "Rb⁺ uptake experiments" to analyze Rb⁺ uptake with Atomic Absorption Spectrophotometry.
12. Perform offset current subtraction and evaluate the time integral of the pump current using the Clampfit program of the pCLAMP package.

9. Rb⁺ Uptake Measurements

1. Cut and melt yellow pipette tip on a 200 μl pipette to about 2 mm diameter. Usage of such tips ensures minimal transfer of solution when the oocytes are moved between incubation or washing dishes. Briefly melt the tip with the flame of a pocket lighter. This polishing step prevents oocyte damage.
2. Prepare for each oocyte to be measured a 1.5 ml Eppendorf tube filled with 1 ml Millipore water. Arrange three Petri dishes with Rb⁺-free washing buffer (e.g. PLB) and one with Millipore water.
3. Transfer five preincubated oocytes simultaneously into a 3.5 cm Petri dish filled with Rb⁺ flux buffer (concentrations from 0 to 15 mM Rb⁺) and incubate for 3 min (Na⁺,K⁺-ATPase) or 15 min (H⁺,K⁺-ATPase) under temperature control (e.g. 21 °C in an air-conditioned room or on a heating plate).
4. Transfer oocytes simultaneously into the first dish with Rb⁺-free washing buffer (in case of Na⁺,K⁺-ATPase PLB can be used). It is mandatory to keep the deviations between the incubation times smaller than 5 sec. Rinse gently.
5. Move oocytes into the second and third dish with Rb⁺-free washing buffer, rinse gently.
6. Transfer oocytes into the dish with Millipore water, rinse gently.
7. Transfer each oocyte individually into one of the prepared Eppendorf tubes filled with 1 ml Millipore water.
8. Homogenize oocyte by pipetting up and down with a 200 μl pipette tip until solution is homogenously turbid.

10. Preparation of AAnalyst 800 Utilizing the THGA Furnace System

1. Turn on valve of the required inert gas (argon) and insert an appropriate hollow cathode lamp (single-element HCLs for Li⁺ or Rb⁺; Photron, Melbourne, Australia) in arbitrary socket position.
2. Start WinLab32 control software (Perkin Elmer) and switch on the appropriate hollow cathode lamp. Perkin Elmer Lumina hollow cathode lamps are recognized automatically and appear on the selection list. Non-compatible hollow cathode lamps need to be entered manually.
3. Allow for a warm-up time of approximately 10-15 min for equilibration of the lamps.
4. Ensure that the THGA tube (transversely-heated graphite atomizer tube, Perkin Elmer, #B3000641) is in good order by visual inspection. THGA furnaces should be replaced every 300 shots (average life-time stated by the manufacturer), but may well endure larger sample numbers.
5. Select the "THGA furnace" technique in WinLab32.
6. Prepare at least five calibration samples with Rb⁺/Li⁺ concentrations between 10 and 50 μg/L (e.g. by appropriate dilution from a 1 M RbCl or LiCl solution) and one blank probe with Millipore water only. Note: Salts should be of highest available purity ("puriss." or from commercially available calibration standards for AAS purpose).
7. Choose the appropriate initialisation file (termed "method" file, which provides a reference to calibration parameters and loads instrument settings, e.g. the AAS detection wavelength, the wavelengths for Rb⁺ and Li⁺ are 780 nm and 670.8 nm, respectively) then enter the positions and concentrations of calibration samples. For this purpose, select the appropriate autosampler (sample tray with 88 or 148 sampling positions for the AAnalyst 800 device).
8. Measure calibration curve.
9. Now transfer each oocyte homogenate into individual 1.2 ml polypropylene sample cups (Perkin Elmer, #B0510397) and place these test tubes into the autosampler tray.
10. Create a sample information file to identify the probes in the autosampler positions, then adjust sample volume to 20 μl, and start the instrument.
11. After completion of the run (or during visual inspection every hour for long series of samples), check for probes in which the measured amount of Rb⁺ exceeds the maximum of the calibration curve. If so, dilute the respective samples appropriately and measure them again.

11. Remarks for Measuring Large Sample Numbers (Above 50)

• For an overnight run of the instrument, it is recommended to fill the reservoir of the autosampler with Millipore water to prevent evaporation of homogenates in test tubes.
• It is recommended to place between each 20 samples a test tube with 2% HNO₃ to prevent clogging of the autosampler's injection capillary.

Recommended THGA-AAS temperature protocol for rubidium

Step	Temperature (°C)	Ramp Time (sec)	Hold Time (sec)	Argon Gas Flow (ml/min)
Dry 1	110	1	20	250
Dry 2	130	5	30	250
Pyrolysis	300-600	10	20	250
Atomization	1700-1800	0	5	0
Clean-out	2400	1	2	250

Recommended THGA-AAS temperature protocol for lithium

Step	Temperature (°C)	Ramp Time (sec)	Hold Time (sec)	Argon Gas Flow (ml/min)
Dry 1	110	1	30	250
Dry 2	130	15	30	250
Pyrolysis	900	10	20	250
Atomization	2200	0	5	0
Clean-out	2450	1	3	250

Data analysis

1. For each measuring condition, calculate mean Rb⁺ uptake. The AAS spectrophotometer yields values in μg/L, which need to be converted into pmol/oocyte/min.
2. For kinetic analyses (apparent KM values), subtract background Rb⁺ uptake determined from uninjected control oocytes from the same batch at the given RbCl concentration.
3. Plot with Origin software, apply fit of a Hill or Michaelis-Menten function to the data. In these equations, [S] is the substrate concentration, nH the Hill coefficient, and K_0.5 the substrate concentration needed for half-maximal activation.

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Результаты

Quantification of Rb⁺ uptake by K⁺- (or Rb⁺)-countertransporting P-type ATPases by AAS in the Xenopus oocyte expression system permits reliable determination of enzyme kinetic parameters.

Determination of the transport stoichiometry of the Na⁺/K⁺-ATPase

For the electrogenic Na⁺/K⁺-ATPase, Rb⁺ fluxes can be determined in two-electrode voltage-clamp experiments aimed at the c...

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Обсуждение

The described method to measure the amount of Rb⁺ (or Li⁺) taken up into individual Xenopus oocytes expressing Na⁺,K⁺- or H⁺,K⁺-ATPase has proven to be a versatile, flexible and accurate technique to determine the kinetic or thermodynamic parameters of transport for cation-countertransporting P-type ATPases^1,4,5,7,8. It is a safe and reliable alternative to radioactive tracer flux assays, and allows addressing a large scope of experimen...

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Материалы

Name	Company	Catalog Number	Comments
4.0 Ethicon Vicryl suture material	Johnson Johnson	V633H
Collagenase type 1A from Clostridium hystolyticum	Sigma Aldrich	C9891
High-Pure PCR Product Purification Kit	Roche Applied Science	11732676001
Nuclease-free water	Ambion	AM9937
mMessage mMachine Kit SP6/T7	Ambion	1340, 1344
Ouabain octahydrate	Sigma Aldrich	O3125
Tricain (ethyl 3-aminobenzoate methanesulfonate salt)	Sigma Aldrich	A5040
Trypsin inhibitor type III-O from chicken egg white	Sigma Aldrich	T2011
SCH28080	Sigma Aldrich	S4443
AAnalyst 800	Perkin Elmer	0993-5256
WinLab32TM	Perkin Elmer
BioPhotometer	Eppendorf	6131 000.012
Borosilicate Capillaries	Science Products	GB150F-8P
Hematocrit tubes 3.5"	Drummond Scientific	3 000-203-G/X
Hollow Cathode Lamp Lithium	Photron	P929LL
Hollow Cathode Lamp Rubidium	Photron	P945
Micropipette Puller	Narishige	Model PC-10
Oocyte Recording Chamber RC-10	Warner Instr.	W4 64-0306
Nanoject II Injection Pump	Drummond Scientific	3-000-204
pCLAMP software	Molecular Devices
Polypropylene Sample Cup (1.2 ml)	Perkin Elmer	B0510397
Speedvac - Concentrator model 5301	Eppendorf	5301 000.210
THGA Tube	Perkin Elmer	B3000641
Turbo TEC-10CX Amplifier	NPI Electronics	TEC-10CX