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.
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.
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/mol1-3, 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 transport4-6, the influence of extra- and intracellular pH7 and the effect of mutations of residues involved in cation coordination during transport4,8. 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.
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
3. In Vitro cRNA Synthesis
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).
5. Enzymatic Isolation of Individual Oocytes
6. cRNA Injection
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:
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:
8. Electrophysiology
9. Rb+ Uptake Measurements
10. Preparation of AAnalyst 800 Utilizing the THGA Furnace System
11. Remarks for Measuring Large Sample Numbers (Above 50)
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
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 correlation between net charge transport (time integral of pump current) and Rb+ transport. Figure 1A shows the recording of pump current (about 40 nA stationary current amplitude) induced by perfusion of a Na+,K+-ATPase-expressing oocyte with a solution containing 1 mM Rb+. Integration of the current signal (grey shaded area under the current trace) yielded 7839 nC of total transported charge (equivalent to 0.0822 nmol of charge). When the same oocyte was subsequently subjected to the AAS determination method, a total amount of 0.176 nmol of Rb+ was found. The ratio between total charge and Rb+ flux was 0.47, which is close to 0.5, the value expected from the 3:2 Na+/K+(Rb+) transport stoichiometry of the Na+,K+-ATPase (net outward transport of one elementary charge corresponds to the uptake of two Rb+/K+ ions). Repetition of such a single-cell experiment several times (Figure 1B) yields a good linear correlation between total charge and Rb+ transport, with a slope factor of 0.49. Thus, even on a single cell level, the AAS technique yields excellently reliable results.
Rb+ flux experiments for analysis of H+/K+-ATPase
For the electroneutrally operating H+/K+-ATPase, the [Rb+] dependence of Rb+ uptake and its sensitivity towards the specific inhibitor SCH28080 can be determined. The data listed in Table 1 have been acquired in a set of three experiments on different batches of oocytes, in which Rb+ fluxes (15 min flux time) mediated by H+,K+-ATPase were measured in Na+-free buffers at pHex 5.5 for different external Rb+ concentrations. Unspecific uptake into uninjected oocytes is less than 10% of the maximally detected Rb+ uptake, and this is insensitive to the addition of SCH28080, whereas the fluxes in H+,K+-ATPase-expressing oocytes are reduced to background level upon addition of 10 μM SCH28080 (at pHex 5.5). Subtraction of the non-specific Rb+ uptake (from uninjected oocytes) yields data as shown in Figure 2. Black squares in Figure 2 refer to the data from Table 1, whereas the open squares were acquired in a similar set of experiments, in which the Rb+ fluxes were determined in Na+-containing buffers. These data yield (1) values for the apparent affinities of H+,K+-ATPase turnover transport for Rb+, and (2) how the apparent affinity is modified by extracellular Na+, showing that extracellular Na+ ions compete for the extracellular facing cation binding pocket during transport, which leads to a decrease in the apparent affinity for Rb+ 4.
The combination of Rb+ uptake experiments under membrane potential control by TEVC and AAS determination can also be applied to determine the voltage-dependence of H+/K+(Rb+) turnover transport under different pH conditions. Data originating from such a combined experiment are listed in Table 2 showing that Rb+ transport by H+,K+-ATPase is only weakly affected by changing the membrane voltage from about -10 to -100 mV. Furthermore, the data in Table 2 show that Rb+ transport by H+,K+-ATPase is stimulated by about a factor of 2 at pHex 5.5 compared to pHex 7.4. On the first sight, this result is counter-intuitive, since an increase in the extracellular H+ concentration should rather lead to a decreased turnover rate because the enzyme has to pump against a steeper H+ gradient. However, it could be shown by additional experiments that a controlled decrease of the cytoplasmic pH by about 0.5 units had the same stimulating effect on transport, irrespective on extracellular pH7. This stimulation is due to a slight acidification of the oocytes’ interior upon incubation in a solution with pHex 5.57. Further information to be drawn from Table 2 is that the addition of 10 μM SCH28080 inhibitor causes different levels of inhibition at pHex 7.4 and 5.5. This is due to the fact that only the protonated form of SCH28080 is pharmacologically active. At pHex 5.5, 50% of the compound is in the protonated, active form, whereas at pHex 7.4, it is only 1%, which leads to a much weaker inhibition7.
Rb+ flux experiments to investigate functional effects of H+/K+-ATPase mutations
Another example for the achievement potential of the AAS detection technique is shown in Figure 3. Figure 3A compares the Rb+ fluxes for several H+,K+-ATPase constructs in which one (ΔY), two (ΔYY), or five (ΔQELYY) amino acids at the C-terminus of the H+,K+-ATPase α-subunit were deleted. For Na+,K+-ATPase, it has been shown that the C-terminus is a distinct structural element, which is critical for structural stabilization of the cation binding sites 14-16. The deletion of the two C-terminal Tyr residues (ΔYY) or of the last five amino acids (ΔKETYY) dramatically affected voltage-dependent parameters of cation transport and led to the occurrence of H+– and Na+-stimulated stationary inward currents in the absence of extracellular K+ 17-19. In order to elucidate whether C-terminal truncations also affect cation transport by H+,K+-ATPase, we assayed Rb+ transport activity of H+,K+-ATPase wild-type and C-terminal truncation constructs as shown in Figure 3A. These data show that Rb+ uptake activity gradually decreases with the extent of the C-terminal deletion. Moreover, the extent of Rb+ uptake inhibition by 10 μM SCH28080, which is high for the WT enzyme, is very much reduced for the ΔY construct and essentially absent for constructs ΔYY and ΔQELYY. At this point, it can not be directly concluded that the decrease in specific Rb+ uptake activity is due to a characteristic effect of the mutations on the transport turnover rate, since also other explanations have to be taken into account, such as general effects on protein folding, stability, or plasma membrane targeting. To correlate the measured Rb+ fluxes with the amount of H+,K+-ATPase protein in the plasma membrane, we prepared plasma membrane (PM) fractions and total cellular membrane fractions (TM) from oocytes expressing the constructs from Figure 3A, and subjected the samples to SDS-PAGE (https://www.jove.com/video/758/electrophoretic-separation-of-proteins)20 and Western blotting with antibodies directed against the H+,K+-ATPase α-subunit. This plasma membrane purification procedure has first been described by Kamsteeg and Deen21 and, later, with some modifications in4,22. Figure 3B, lower panel, shows that the relative amounts of total H+,K+-ATPase α-subunit protein in the TM fraction are very similar for all H+,K+-ATPase constructs investigated. However, in the PM fraction (Figure 3B upper panel), the relative amount of protein also gradually decreases with increasing extent of the C-terminal deletion. Therefore, one can conclude from this combination of experiments that truncations at the C-terminus of the H+,K+-ATPase’s α-subunit affect plasma membrane expression indicating that folding and structural stability of the α-subunit or association with the β-subunit might be affected by the truncations. This also serves as an explanation for the reduction of specific Rb+ uptake. However the SCH28080 inhibition data yield additional information for the truncation constructs. The SCH28080 inhibitor targets a specific conformational substate of the catalytic cycle, the so-called E2(P) conformation (P denotes a phosphorylated intermediate) with cation binding sites oriented towards the extracellular fluid and with high affinity for Rb+/K+. Substantially reduced sensitivity of the C-terminally truncated proton pumps towards 10 μM SCH28080 in the Rb+ uptake experiments (black bars in Figure 3A) suggests that the steady-state distribution of reaction cycle intermediates is shifted to E1(P)-like conformational substates, in which the cation binding pocket is exposed to the intracellular medium4. These data also show that it is important to correlate functional with biochemical data, if profound mechanistic conclusions are desired.
Temperature dependence of Rb+ fluxes
Rb+ fluxes can also be utilized to measure thermodynamic parameters like activation energies. As shown in Figure 4A, Rb+ uptake by H+,K+-ATPase-expressing oocytes is strongly dependent on temperature. Notably, even at 34 °C incubation temperature during Rb+ uptake, non-specific Rb+ uptake is still low, as can be seen from the low value of the flux at 34 °C in the presence of the SCH28080 inhibitor. Plotting the decadic logarithm of the Rb+ uptake against the reciprocal temperature (Arrhenius plot) yields a good linear correlation of the data, resulting in an activation energy of 96 kJ/mol from the slope of the curve, a value, which is typical for enzyme-catalyzed reactions and also agrees well with values determined for pump currents of Na+,K+-ATPase measured in the Xenopus oocyte expression system1.
Li+ uptake measurements
Besides Rb+, also Li+ uptake by H+,K+-ATPase can sensitively be determined by the AAS technique. Figure 5 shows that unspecific Li+ uptake in uninjected oocytes is quite low, and that uptake increases for H+,K+-ATPase-expressing oocytes with increasing Li+ concentrations up to 60 mM. This is different for Rb+, for which saturation of Rb+ uptake is observed already at concentrations above 5 mM. Notably, Li+ uptake activity is larger than Rb+ uptake activity. Furthermore, 10 μM SCH28080 hardly reduces Li+ uptake (compare bars for 30 mM Li+ with and without inhibitor), whereas in the case of Rb+, inhibition is stronger. Notably, this experiment has been carried out at pHex 7.4, at which only a small fraction of the SCH28080 compound is in the active (protonated) form. Thus, Li+ could also act in terms of inducing preference of the enzyme towards E1(P) state(s)7 during stationary H+/Li+ turnover cycling.
[Rb+] / mM | normalized Rb+ flux for HKα-S806C + HKβ | |
no inhibitor | + 10 μM SCH28080 | |
5 (uninjected oocytes) | 0.07 ± 0.01 | 0.074 ± 0.002 |
0.05 | 0.17 ± 0.01 | 0.05 ± 0.02 |
0.1 | 0.31 ± 0.01 | 0.05 ±0.03 |
0.25 | 0.50 ± 0.03 | 0.05 ± 0.03 |
0.5 | 0.64 ± 0.03 | 0.07 ± 0.04 |
1 | 0.80 ± 0.03 | 0.08 ± 0.05 |
2.5 | 0.83 ± 0.06 | 0.15 ± 0.05 |
5 | 1.00 ± 0.05 | 0.13 ± 0.04 |
Normalization value: mean Rb+-flux (at 5 mM Rb+) from 3 separate experiments, 29.2 pmol/oocyte/min (10-15 oocytes per data point in each experiment) |
Table 1. SCH28080 sensitivity of Rb+ uptake by oocytes expressing HKα-S806C/HKβ. Rb+ uptake was determined at pH 5.5 in extracellular Na+-free solution containing different Rb+ concentrations in absence or presence of 10 μM SCH28080. Data were normalized to Rb+ uptake at 5 mM [Rb+] and are given as means ± S.E.M. from 10-15 oocytes out of a single batch.
pHex7.4 (in pmol/ooc./min) | pHex 5.5 (in pmol/ooc./min) | |
Vm = -100 mV | 11.9 ± 1.1 | 24.5 ± 1.3 |
unclamped (-10…-20 mV) | 13.9 ± 1.5 | 26.5 ± 1.2 |
control: pHex 5.5, -100 mV + 10 μM SCH28080 | 2.4 ± 0.8 |
Table 2. Voltage dependence of Rb+ uptake by oocytes expressing HKα-S806C/HKβ. Values represent Rb+ uptake (in pmol/oocyte/min) at 5 mM Rb+ and pHex 7.4 or 5.5 for oocytes expressing HKαS806C/βWT, which either were held at -100 mV by two-electrode voltage-clamping, or subjected to Rb+ uptake without voltage control (the “resting” membrane potential was measured to be between 10 and -20 mV, as determined independently). Data are means ± S.D. from oocytes of one cell batch (N=12…23).
Figure 1. Correlation of Rb+ uptake by Na+,K+-ATPase with pump current in two-electrode voltage-clamp (TEVC) experiments. (A) Stationary pump current recording at -10 mV membrane potential measured on an oocyte expressing ouabain-resistant human Na+,K+-ATPase α2/β1-subunit complexes. The pump current was initiated by solution exchange from a Rb+-free to a 1 mM Rb+-containing solution (white and hatched bars above current trace, respectively). The time integral of the current (here: 7840 nC) corresponds to a net outward transport of 0.082 nmol of elementary charges. When the measured oocyte was removed from the recording chamber and subjected to the AAS procedure, a total amount of 0.176 nmol Rb+ was found in the cell, which is about 2.1 times the amount of transported charge. (B) Correlation between total transported charge (time integral of pump current) measured in two-electrode voltage-clamp Rb+ flux experiments on single oocytes with the amount of Rb+ per cell measured subsequently by AAS. The graph indicates that even with single cells, a good linear correlation (see values for the reduced χ2 and correlation factor R2) between charge and Rb+ uptake can be obtained. The slope factor derived from a linear fit to the data (0.49 ± 0.10) is consistent with the uptake of 2 Rb+ ions per transported charge, in agreement with the 3Na+/2K+(Rb+) transport stoichiometry of the Na+,K+-ATPase.
Figure 2. Concentration-dependent Rb+ uptake by H+,K+-ATPase. Normalized Rb+ uptake in the absence (filled squares) or presence (open squares) of extracellular Na+ at pH 5.5 is depicted. Oocytes were injected with cRNA for the HKα-S806C mutant and the HKβ-subunit. Data were normalized to Rb+ uptake at the maximal RbCl concentrations used, with the following values (in pmol/oocyte/min: 29.3, 27.4, 30.8 in absence of Na+; 21.1, 23.4 in presence of Na+). Data are means ± S.E.M., resulting from 8-12 oocytes out of three experiments. Apparent half-maximal activation constants K0.5 were obtained from a fit of a Michaelis-Menten function to the data (fit curves shown as dashed and dotted line, respectively).
Figure 3. Rb+ uptake by C-terminally truncated H+,K+-ATPase constructs. (A) H+,K+-ATPase-mediated Rb+ uptake at 5 mM RbCl in the absence (hatched bars) or presence (black bars) of 10 μM SCH28080. Results from uninjected control oocytes, oocytes injected with cRNA of the HKβ-subunit and either HKα-S806C or HKα-constructs with the indicated C-terminal truncations are shown. Data are means ± S.E.M. from 3 individual experiments with 15-20 oocytes, normalized to Rb+ uptake of the HKα-S806C/HKβ (corresponding to 20.4, 23.7 and 29.5 pmol/oocyte/min, respectively). (B) Western blot analysis of plasma membrane (PM, upper panel) and total membrane fractions (TM, lower panel) isolated from H+,K+-ATPase-expressing oocytes. Detection used anti-HKα-subunit antibody HK12.1823. One representative Western blot out of at least 3 from different oocyte batches is shown.
Figure 4. Temperature dependence of Rb+ uptake by gastric H,K-ATPase. (A) H+,K+-ATPase-mediated Rb+ uptake (in pmol/oocyte/min) at 5 mM Rb+ and pHex 5.5 (light gray bars) at temperatures between 18 and 34 °C, as indicated. White bars represent Rb+ uptake of non-injected control oocytes at each temperature under the same conditions. The gray bar at 34 °C shows Rb+ uptake at pHex 5.5 in the presence of 10 μM SCH28080 inhibitor. Data in each column are means of 20-25 oocytes from oocytes of one cell batch. (B) Arrhenius diagram (plot of the decadic logarithm of Rb+ uptake versus the reciprocal temperature) for temperature-dependent Rb+ uptake from the data in (A). Each data point is given as the mean ± S.E.M. of three independent experiments. For normalization, the Rb+ uptake at 34 °C was taken for each experiment. The activation energy obtained from a fit of a straight line to the data (dashed line) is about 96 kJ/mol.
Figure 5. Comparison of Li+ and Rb+ uptake by H+,K+-ATPase. H+,K+-ATPase-mediated uptake of Li+ at the indicated concentrations of LiCl, and of Rb+ at 5 mM RbCl. The extracellular pH was 7.4. Results from uninjected control oocytes, and of H+,K+-ATPase-expressing oocytes in the presence of a certain [Li+] or [Rb+]and 10 μM inhibitor SCH28080 are also included. Data are means ± S.E.M. from 8-15 oocytes of a single batch.
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 ATPases1,4,5,7,8. It is a safe and reliable alternative to radioactive tracer flux assays, and allows addressing a large scope of experimental questions within considerably short time. In accordance, only very limited data have been acquired with radiotracers24-27. The AAS method is particularly useful for H+,K+-ATPase, which – due to its electroneutral transport activity – cannot be analyzed by standard electrophysiology. However, the exemplary data shown in this protocol demonstrate that the combination of flux measurements with voltage-clamping can even resolve the voltage dependence of electroneutral transporters. From our experience, it is sufficient to employ flux times of 3 min (for Na+,K+-ATPase) or 15 min (for H+,K+-ATPase). These times could easily be reduced by adjusting the volume of fluid used to homogenize individual oocytes after uptake.
We consider the following points as crucial for the technique:
The described technique is not limited to Rb+ and Li+ transport, but could easily be extended to other trace elements, especially transition or heavy metals of biological relevance, for which AAS hollow cathode lamps are available, such as Cs, Mn, Mo, Fe, Co, Ni, Cu, or Zn. Furthermore, the method is not limited to K+-countertransporting P-type ATPases. Essentially any metal ion transporter that can be expressed in the plasma membrane of Xenopus oocytes, and even endocytotic uptake mechanisms can in principle be investigated. In the special case of H+,K+-ATPase, which is not accessible by standard electrophysiology, the AAS flux method has provided deep insight into the underlying transport kinetics and structure-function relationships.
Of note, the technique also supports pharmacological screening. The gastric H+/K+-ATPase is an important therapeutic target for drugs known as acid suppressants or proton pump inhibitors. While there is still a strong need for improved compounds with better bioavailability, pharmacokinetics, or less side effects, only very few assays are available that meet the strict legal approval regulations. Although the THGA-AAS method with Xenopus oocytes will not allow for high-throughput screening, it is excellently suited for profiling e.g. hundreds of derivatives of promising lead structures with high information content and in reasonably short time.
Due to the high relevance of Na+ in natural transport processes across membranes, a similarly sensitive detection of Na+ would be highly desirable. Although we paid considerable effort on determining Na+ by THGA-AAS, the element has proven to be very problematic, since Na+ is a frequent and ubiquitous environmental contaminant. Even the preparation of absolutely Na+-free reference buffers in laminar flow benches did not lead to reliable results, since our AAS device is not installed in a clean room. Thus, in the case of Na+, other experimental approaches such as inductive coupled plasma emission spectroscopy (ICP-ES) might give better results.
The authors have nothing to disclose.
The authors thank Ernst Bamberg (Max-Planck-Institute of Biophysics, Frankfurt, Germany) for generous support during the initial phase of method development, Kazuhiro Abe (Kyoto University, Japan) for numerous fruitful discussions and Dr. Michael Kohl (Analytik-Service, Woltersdorf, Germany) for technical support. The authors gratefully acknowledge funding by the German Research Foundation DFG (Cluster of Excellence “Unifying Concepts in Catalysis”), which also financed the Perkin Elmer AAnalyst 800 apparatus (SFB 498).
Name of the Reagent | Company | Catalogue Number | Comments (optional) |
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 |