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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

Groupe d'étude des protéines membranaires (GÉPROM), and Département de physique, Université de Montréal, Montréal, Québec, Canada

Submitted 22 November 2007 ; accepted in final form 24 September 2008

	ABSTRACT

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The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na⁺/glucose cotransporter (SGLT1) using Na⁺- and H⁺-selective electrodes and with the electroneutral H⁺/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na⁺/glucose influx, which generated a measurable decrease in the trap Na⁺ concentration ([Na⁺]_T) by 0.080 ± 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na⁺]_T, which was proportional to the increased cotransport current. In a Na⁺-free, weakly buffered solution (pH 5.5), H⁺ drives glucose transport through SGLT1, and this was monitored with a H⁺-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na⁺]_T or [H⁺]_T were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na⁺]_T or [H⁺]_T is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.

ion-selective electrode; cotransporter; SGLT1; MCT1; electrogenic; electroneutral; transporters

	MATERIALS AND METHODS

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ISE fabrication. Borosilicate capillaries (1-mm outer diameter x 0.5-mm inner diameter with fibers; FHC, Bowdoin, ME) were pulled using an horizontal puller (Flaming/Brown P-97; Sutter Instruments, Novato, CA), and the tips were broken with forceps, while visualized under a microscope, to provide an approximate tip diameter of 20–100 µm; the tips were then fire polished to create a smooth surface. Larger tip diameters could not be reliably used, as the ion-selective resins were not stably retained within the tip of these ISEs on retracting the ISE from the oocyte surface, which resulted in significant drift in the signal. The microelectrode was briefly dipped in a 1:200 mixture of 90% dichlorodiphenylsilane (Sigma-Aldrich, Oakville, ON, Canada) and 99.9% acetone (Sigma-Aldrich) and then positioned tip-up on a rack and baked at 120°C for an hour. Before use, the electrode was backfilled with a small volume (<1 mm of capillary length) of ion-selective resin using thin capillary tubing and a syringe. Unused silanized electrodes can be stored in a desiccator or in an oven set at 80°C for days without significantly affecting their properties.

Na⁺-selective electrodes were made with a Na⁺-neutral carrier cocktail (cat. no. 71178; Fluka, Oakville, ON, Canada) and were backfilled with 100 mM NaCl. According to the manufacturer, the resin provides an ideal Nernstian response down to 3.2 mM (2), presents a weak Na-to-K permeability ratio (P_Na/P_K = 2.5), but is insensitive to changes in Ca²⁺ concentration. In our hands, the performance of large-diameter Na⁺ electrodes was tested by immersing the ISE in a solution containing, in mM, 100 NaCl, 3 KCl, 0.82 MgCl₂, 0.74 CaCl₂, 10 HEPES and adjusted to pH 7.6 with Tris. Na concentration was reduced to 50, 20, 10, 5, and 2 mM by replacing Na by N-methyl-D-glucamine (NMDG). The signal from a bath KCl flowing electrode (1 M) was subtracted from the ISE signal to correct for minor changes in liquid junction potentials at the bath agar bridge. For eight different ISEs, the average slope of the electrode signal as a function of log ([Na]) was 51.3 mV/decade between 20 and 100 mM and was slightly reduced to 48.5 mV/decade between 5 and 20 mM (Fig. 1A). Starting from a [Na] of 10 mM, replacing NMDG with choline had no effect, but replacing 80 mM NMDG by 80 mM K produced a positive shift of the ISE signal by 32 mV (n = 6). This is consistent with P_Na/P_K of 2.03. Sodium-selective electrodes were completely insensitive to the presence of 5 mM -methyl-D-glucose (MG) or 200 µM phlorizin (Pz), the substrate and specific inhibitor of SGLT1, respectively.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Calibration curves for Na+- and H+-selective electrodes are shown. A: average signal from Na+-selective electrodes (VNa; mean ± SD, n = 8, open circles) as a function of Na+ concentration (N-methyl-D-glucamine replacement). The data represented by open triangle are the electrode signals in the presence of 10 mM Na+ and 80 mM K+ (mean ± SD, n = 6). The slope of the straight line between 5 and 20 mM Na is 48.5 mV/decade. B: average signal from H+-selective electrodes (VH; mean ± SD, n = 6, open diamonds) as a function of pH. The slope of the line between pH 5.5 and 7.5 is 56.8 mV/pH unit.

Oocyte preparation. Oocytes were surgically removed from X. laevis frogs, dissected, and defolliculated as previously described (10). Healthy oocytes were injected with 46 nl of water containing 0.1 µg/µl mRNA coding for human myc-hSGLT1 [as previously demonstrated (3), this epitope-tagged version of human SGLT1 displays properties that are indistinguishable from the untagged form] or 0.1 µg/µl mRNA coding for rat electroneutral H⁺/monocarboxylate transporter (MCT1; kindly provided by Dr. Andrew Halestrap, University of Bristol). Oocytes were maintained in Barth's solution [in mM: 90 NaCl, 3 KCl, 0.82 MgSO₄, 0.41 CaCl₂, 0.33 Ca(NO₃)₂, 5 HEPES, pH 7.6] supplemented with 5% horse serum, 2.5 mM Na⁺ pyruvate, 100 U/ml penicillin, 1 mg/ml kanamycin, and 0.1 mg/ml streptomycin for at least 3 days following injection before electrophysiological experiments were performed.

Solutions. When needed, MG (a nonmetabolized glucose analog) and/or 200 µM Pz were added to the Na⁺ buffer (in mM: 90 NaCl, 3 KCl, 0.82 MgCl₂, 0.74 CaCl₂, 10 HEPES and adjusted to pH 7.6 with Tris). Sodium replacement was performed with NMDG. Sodium-free acidic solutions (pH 5.5) were buffered with 1.5 mM MES. In Na⁺-free, acidic solutions, the affinity for MG is greatly reduced (K_m^MG = 20 mM at –50 mV; Ref. 20), and 35 mM MG was needed to obtain a large cotransport current. Under these conditions, 35 mM D-mannitol was present in the pH 5.5 solution to prevent osmotic shock when switching to the high MG concentration solution; for both solutions, the NaCl (or NMDGCl) concentration was reduced to 65 mM (50 mM mannitol replacement). For studies employing the electroneutral H⁺/MCT1, a Na⁺ buffer with 1 mM MES (pH 6.5) was used. Unless otherwise mentioned, all chemicals were obtained from Sigma-Aldrich.

Recording circuitry. Voltage-clamp recordings and current filtering (1 kHz) were performed using the OC-725C two-electrode voltage-clamp headstage and amplifier (Warner Instruments, Hamden, CT). Data recording was performed using a Digidata 1322A recording system and pCLAMP 8.2 software (Axon Instruments, Union City, CA). The microelectrodes were filled with 1 M KCl and presented resistances <2 M. Ion-selective measurements were performed with the Duo 773 dual-channel, high-impedance electrometer (World Precision Instruments). The signal from a supplementary 1 M KCl microelectrode placed near the ISE was automatically subtracted from the ion-selective signal to eliminate the voltage drop associated with the bath series resistance. The ion-selective signal (V_ion) was amplified by the electrometer, digitized, and recorded unfiltered by pCLAMP. A grounded piece of aluminum foil was wrapped around the ISE holder to reduce electrical noise.

Pulse protocols and data analysis. A variety of voltage pulse protocols were used with voltages ranging from –155 mV to 40 mV. In all cases, the holding potential was set at –50 mV. When needed, 10–200 identical pulses were averaged by pCLAMP to reduce electrical noise in V_ion. Extra filtering was achieved using the Clampfit low-pass Bessel filter [cutoff frequency (F_c) = 100 Hz]. Charge transfer curves were obtained as previously described (10). Briefly, current in the presence of Pz was subtracted from currents measured in the Na⁺ buffer lacking Pz. The resultant signal was corrected for steady-state currents by subtracting the mean of a 20-ms time window positioned at the end of the pulse. Integrating this signal yielded the total charge transferred at each potential. Fast V_ion changes were obtained by averaging 20-ms time windows at 75 or 125 ms after the initiation of the pulse. Slopes were obtained by fitting a straight line to the linear part of the V_ion vs. time curve. For steady-state measurements of V_ion, a slow linear drift correction was applied to make sure that identical solutions yielded identical V_ion at the two different times. This drift correction was measured and applied in each individual experiment; its amplitude averaged –0.7 ± 1.2 µV/s (n = 9). Typical recordings are presented, and similar experiments have been repeated three times or more in oocytes obtained from at least two different donors. All statistical results represent means ± SE.

	RESULTS

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A X. laevis oocyte expressing hSGLT1 was transferred to the bath of a classical two-electrode voltage clamp setup. Current and voltage electrodes were inserted in the oocyte, and the membrane potential (V_m) was clamped at –50 mV. With the use of a third micromanipulator, the tip of the ISE was immersed in the bath solution and calibrated using extracellular Na⁺ concentrations of 90 and 10 mM or using Na⁺-free solutions adjusted to pH 7.5 and 5.5, depending on the selectivity of ISE used. The ISE was then gently pushed against the oocyte vitelline membrane, and a fourth manipulator was used to position a reference KCl electrode within 50 µm of the ISE tip. At this point, two different types of measurement can be performed. To examine steady-state conditions, the trap ionic concentration is measured over time courses of several min as a membrane transport mechanism is stimulated by addition of a cotransported substrate to the bath (this portion of the procedure can be applied to electroneutral transport systems) or by a change in the oocyte holding potential. The ionic concentration measured corresponds to the trap ionic concentration needed to balance the ionic flux through the membrane patch bounded by the ISE with the ionic diffusion across the seal formed at the ISE/membrane junction. To examine initial rate conditions (for electrogenic membrane transport systems only), the transport system is abruptly stimulated by a voltage pulse, and the initial change in the trap ionic concentration is measured. In this case, the ionic gradient between the trap and the bath solution remains almost constant, and the change in the trap ionic concentration reflects exclusively the change in the ionic transport rate in the membrane patch bounded by the ISE.

Steady-state concentration measurements. Figure 2A presents a typical experiment where a Na⁺-selective electrode was applied against the surface of a voltage-clamped oocyte expressing hSGLT1. The external solution was switched from a normal saline solution, containing 90 mM Na⁺, to a solution where Na⁺ had been reduced to 10 mM using NMDG as the replacement cation. As expected, the Na⁺-selective signal (V_Na) became more negative and, after 100 s, stabilized to a level that was 53 mV more negative than the value recorded in the presence of 90 mM Na⁺. Adding 5 mM MG to the bath solution was associated with a total oocyte current change of –205 nA. A drop in the trapped sodium concentration ([Na⁺]_T) could be simultaneously observed from 10.11 to 10.03 mM (Fig. 2A). In a series of eight experiments performed on five different oocytes, we found that, on addition of MG to the bath solution, an average current of –245 ± 18 nA was generated, and the trap V_Na decreased by an average of 203 ± 22 µV, corresponding to a [Na⁺]_T of –0.080 ± 0.009 mM. Please note that this type of experiment can be performed with an electroneutral transport mechanism. In the experiment shown, the drop in [Na⁺]_T was further increased by changing V_m in 10-mV steps from –50 mV to –90 mV. After the total oocyte current was corrected for the presence of a current, unrelated to SGLT1 (the current in the presence of Pz was measured at each V_m before the experiment), a linear correlation was observed between the change in [Na⁺]_T and the voltage-dependent change in the cotransport current (Fig. 2B). In the absence of MG, the Pz-sensitive current (the so-called leak current) measured at –50 mV is minimal, and [Na⁺]_T reached the value predicted by the linear relationship between the Pz-sensitive current (I_Pz) and [Na⁺]_T.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Steady-state trapped sodium and pH measurements ([Na+]T, pHT) are shown. A: the solution was first switched from normal saline (90 mM Na+) to a 10 mM Na+ solution. Then, 5 mM -methyl-D-glucose (MG) was added to the 10 mM Na+ buffer at –50 mV. After reaching a steady [Na+]T measurement, membrane potential (Vm) was stepped from –50 to –90 mV in 10-mV increments. Vm was then stepped back to –50 mV, and MG was removed. The electrode was calibrated by pulling the electrode from the oocyte surface to measure the true 10 mM value (data not shown). B: correlation between [Na+]T and MG current (IMG). The substrate was added at Vm = –50 mV, and then the voltage was stepped, in 10-mV increments, to –90 mV. All currents in the presence of MG were corrected for the currents measured in the presence of phlorizin (Pz) and were fitted with a straight line. C: steady-state surface pH (pHT) measurement. The solution was switched from pH 7.6 to pH 5.5. When a steady pHT was reached, 35 mM MG was added to the bath. Vm was then stepped to –90 mV in 10-mV increments and returned to –50 mV. MG was removed from the bathing solution, and Pz was added. The ion-selective microelectrode (ISE) was then pulled away from the oocyte to measure the true pH 5.5 value (arrow). Data was reduced to yield 1 point per second. The 3 vertical interruptions in the current trace represent periods where series of voltage pulses were applied to the oocyte. D: comparison of pHT and IMG. Currents in the presence of MG were corrected for the currents measured in the presence of Pz. The substrate and inhibitor were added at –50 mV. Im, membrane current; IPz, Pz-sensitive current.

To illustrate use of the ion-trap technique with electroneutral transporters, a H⁺-selective electrode was used to detect proton fluxes associated with the H⁺/lactate^– cotransporter MCT1 (11). As shown in Fig. 3, a pH-ISE was brought into the vicinity of a nonvoltage-clamped oocyte expressing MCT1. When the ISE was put in contact with the oocyte, the pH signal rapidly moved from 6.52 to 6.62. This effect can also be observed with SGLT1-expressing oocytes as well as with noninjected oocytes and must be due to an endogenous proton influx or to a reaction of the ISE on establishing physical contact with the oocyte vitelline membrane. After 1–2 min, the pH signal was stabilized, and a solution containing 0.2 mM lactate was introduced into the bath. The trap pH increased to 6.72 within 20 s. This alkalinization of the trap reflects a lactate-induced proton influx that is decreased by successively reducing the bath lactate concentration to 0.1 and 0 mM. When the ISE is pulled away from the oocyte, the bath pH was correctly measured, and a calibration was performed (during the recording interruption in Fig. 3). Finally, the constant pH of all solutions used was checked directly with the ISE in the experimental chamber. Addition of 0.1 mM lactate produced alkalinization by 0.093 ± 0.015 pH unit, whereas addition of 0.2 mM lactate produced additional alkalinization by 0.086 ± 0.009 pH unit (n = 5 oocytes). Lactate addition produced no significant effects in measurements using noninjected oocytes (data not shown).

View larger version (7K): [in this window] [in a new window]   Fig. 3. Steady-state trapped pH measurements (pHT) on an oocyte expressing the electroneutral monocarboxylate transporter, MCT1, are shown. At the time indicated by the down arrow, the ISE was put in contact with the oocyte surface. Adding lactate to the weakly buffered extracellular solution, 1 mM MES, pH 6.5, resulted in a concentration-dependent alkalinization of the trap. At the time indicated by the up arrow, the ISE was pulled 50–100 µm away from the oocyte, and the same lactate-containing solutions were introduced in the bath.

Fast [Na⁺]_T measurements.

MG

MG

0MG

(1)

Under these conditions, if Q_dep is set to 5 nC, the best parameter values are: V_0.5 = –164 ± 21 mV, Q_hyp = –50 ± 14 nC, and dV_m = 48 ± 3 mV. The Boltzmann fit of Q shown in Fig. 6B is superimposed onto the Step vs. V_m curve, and it can be seen that the two parameters vary across membrane potentials almost identically with each other. This is consistent with the hypothesis that a rapid change in V_m generates an electrogenic conformational change that can be observed as the Q vs. V_m curve. This conformational change allows the Na⁺ binding site to be exposed to one side or the other of the membrane, and a fraction of the charge movement detected is thought to be associated with the binding/debinding of Na⁺ itself. Using the ion-trap technique, this Na⁺ movement between the trap and the cotransporters can be directly detected.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Initial rate measurements of [Na+]T during voltage pulses are shown. A: the signals presented are averages of 10 series of the voltage pulses shown. Currents and VNa were low-pass filtered at 1 and 0.1 kHz, respectively. Bath Na+ is 10 mM, and pH is 7.6. VNa is shown uncorrected for series resistance artifacts. When 5 mM MG was added to the bath, a slope appeared in the VNa signal. Pz abolished the Na+/glucose cotransporter (SGLT1)-mediated pre-steady-state currents and reduced the amplitude of VNa below the level seen in the Na+ buffer. B: initial rate [Na]T measurement for a noninjected oocyte.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Slope () and current (IMG) comparison for multiple experiments using Na+ electrodes is shown. The slope, during a voltage pulse, is obtained by fitting a straight line to the linear part of VNa and is 0 at –50 mV. The average slopes for 8 experiments are shown with interpolation. The MG-dependent current was obtained by averaging a 20-ms time window at the end of the leak-corrected signal. The mean MG-dependent currents for 8 experiments are shown with interpolation.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Series resistance correction and comparison of charge transfer (Q) and Step (VNa) curves are shown. A: the apparent resistance was obtained by fitting a straight line through the VNa vs. IPz data. These values were obtained at holding potentials ranging from –140 to 40 mV by averaging a 20-ms time window at the end of the pulse (dashed boxes). B: the charge transfer (Q) was obtained by integrating the Pz-sensitive pre-steady-state currents. Q is shown with its Boltzmann fit superimposed (solid line). After the correction of VNa with the apparent resistance obtained as described in A, a 20-ms time window was averaged at the end of the pulse. The average Step derived from 8 experiments is shown on the right with the Boltzmann fit of Q normalized and superimposed (dashed line).

Fast pH_T measurement.

0MG

MG

MG

View larger version (20K): [in this window] [in a new window]   Fig. 7. Simultaneous oocyte current Im and pHT measurements (VH) in a pH 5.5 buffer are shown. A: the signals shown were selected from a series of voltage pulses that were repeated 8 times for averaging purposes. Currents and VH were low-pass filtered at 1 and 0.1 kHz, respectively. VH is shown uncorrected for series resistance artifacts. In the pH 5.5 buffer, a significant pre-steady-state associated pHT (Step) appears with a nonnegligible, leak-associated slope. The signal is completely Pz-sensitive. B: initial rate pH measurements for a noninjected oocyte exposed to 35 mM MG.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Comparison of electrophysiological steady-state and pre-steady-state current measurements with the observations provided by the ion-trap technique is shown. A: average MG-sensitive current (IMG) with the slope () measured in the VH vs. t recording in the presence of 2.5 and 35 mM MG (n = 3). The interpolation of IMG at each concentration (full line) was normalized and superimposed on the VH slope curve (dashed line). B: average charge transfer (Q) obtained from the integration of the pre-steady-state transient currents (n = 13) with its Boltzmann fit superimposed (solid line) and average corrected Step in the VH vs. t recording (n = 18) with the normalized Boltzmann fit of Q superimposed (dashed line).

	DISCUSSION

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Detection limits. Traditionally, ISEs have tip diameters much smaller than 1 µm and present impedances on the order of tens of G (9). They are characterized by a high electrical noise level and slow rise time. To overcome these limitations, we used large ISEs. We quickly found that ISEs with tips >100 µm do not hold resin tightly enough to perform long experiments, whereas tips <20 µm exhibited an unacceptably high resistance (>500 M), which compromised the sensitivity and the time resolution of the technique. We thus settled on ISEs in the 20–100 µm range. We routinely achieve ISE impedances as small as 100 M, thus reducing their characteristic response time to 20 ms (Figs. 4 and 7). More than 70 ISE were fabricated during the time course of the present study, and, in our view, their characteristics are reproducible, and the variability between experiments stems primarily from the size of the trap and the resistance of the seal rather than from the performance of the electrode used. If the signal is filtered with a low-pass Bessel filter with a 1-kHz cut-off frequency, a typical noise level of 0.1 mV can regularly be achieved. When used to detect ion concentration changes within a 150-ms voltage pulse, the cut-off frequency can be reduced to 100 Hz, and the noise level can be lowered to 0.04 mV. The noise level can be further reduced by a factor of 3-to-10 by averaging 10–100 identical voltage pulses. For nonelectrogenic transporters, steady-state measurements must be used, and the time response is limited by the time typically required for the extracellular solution to gain access to the trap (20 s). Under these circumstances, the ion-trap signal can be digitized at 100 Hz, and local averaging can be performed to yield 1 point per second displaying a noise level of 10 µV. The sensitivity of the signal is then limited by spontaneous fluctuations reaching 0.1 mV in amplitude over periods of minutes (Fig. 2A). For an ideally selective ISE, a sensitivity of 0.1 mV corresponds to a change in ionic concentration of 0.4%.

Steady-state measurements. The ion-trap technique was first used to examine steady-state conditions, i.e., measuring the ionic concentration in the trap when the flux across the membrane is exactly balanced by diffusion across the seal resistance between the ISE and the plasma membrane. In the absence of MG, [Na⁺]_T is very close to the bath Na⁺. Addition of glucose generates a shift in V_Na, which can be resolved by this technique as indicated by the small SE associated with this measurement (–0.080 ± 0.009 mM). Note that, under steady-state conditions, the transporter need not be electrogenic, and any transporter mediating a Na⁺ flux of 7 nmol/h in an oocyte would generate the same signal. Employing an electrogenic transporter allowed us to change the ionic flux rapidly through application of different membrane potentials. When V_m is changed from –50 to –90 mV in 10-mV increments, Fig. 2, A and C, as well as Fig. 3 show that <20 s are required for a new equilibrium to be reached between the trap and the bath and that the steady-state value reached after this short delay is proportional to the cotransport current.

Steady-state measurement can also be performed while measuring external pH (pH_T) in the absence of Na⁺. On changing the perfusion solution from a pH 7.5 buffer to a pH 5.5 buffer, pH_T became more acidic but stabilized before reaching pH 5.5. This steady-state difference is due to the presence of a significant leak current mediated by SGLT1 at acidic pH. This is confirmed by the fact that Pz perfusion inhibited the leak current and induced a further acidification of the trap that progressively tended toward bath pH (Fig. 2, B and D). This indicates that the leak current, in the absence of Na⁺, is indeed mediated by proton transport. This is a significant observation as the ionic nature of the leak current through SGLT1 remains poorly understood. Addition of MG generated a large inward current and concomitant alkalinization of the trap. Figure 2D shows that the pH_T, reached at steady state, is proportional to the MG cotransport current. With H⁺ fluxes in the presence of a weakly buffered extracellular solution (1.5 mM MES), the method detection limit would correspond to a change in cotransport current of 20–50 nA, which corresponds to a proton flux of 0.7–1.8 nmol/h per oocyte.

Initial rate measurements. The ion-trap technique can also detect voltage-dependent changes in the trap ionic concentrations over the initial 20–150 ms of a voltage pulse. During this very short period, the ionic concentration in the trap is far from attaining a new steady-state value as 20 s would be required to achieve this. We are thus dealing with initial rate conditions where the change in the trap ionic concentration reflects a change in the ionic transport across the membrane covered by the ISE with minimal changes in the ionic flux across the seal between the trap and the external solution. The changes in V_ion are rather small over such short time periods, and a correction needs to be applied for the voltage drop across the seal resistance. This is best measured in the presence of Pz such that no SGLT1-dependent ion fluxes can occur across the patch of membrane covered by the ISE. Under these conditions, there remained a voltage-dependent change in V_ion that was proportional to the total oocyte current (Fig. 6A). This V is thought not to represent any change in the local cationic concentration since it is still present when the absolute sensitivity of the electrode is decreased by raising the ambient cationic concentration well above detectable limits (90 mM for Na⁺ electrode; data not shown). In eight experiments, the slope of the V_Na vs. I_m (Fig. 6A) averages 100 ± 30 . This is an apparent seal resistance since it is calculated using the total current of the oocyte instead of the current fraction passing through the membrane patch covered by the ISE. Assuming that the ISE covers 1/2,000th of the oocyte surface, this apparent resistance would correspond to an effective seal resistance of 230 ± 70 k. When this ohmic V is subtracted from the ion-sensitive signal, a significant voltage-dependent V_ion remains, which represents a real change in the local cationic concentration. This V_ion is thought to represent the cations that bind to or are released from the cotransporter when a change in membrane potential produces a reorientation of the cation binding site. According to the current SGLT1 transport model, we expect an extracellular change in cation concentration that is proportional to the pre-steady-state charge displacement (as seen in a Q-V_m curve). Our results are in agreement with this idea since the Q-V_m curve nicely reproduces the characteristics of the Step-V_m curve with either Na⁺- or H⁺-selective electrodes (Figs. 6B and 8B).

In the presence of MG, a slope in the V_Na vs. time curve was observed. This slope can be used to estimate the equivalent height (h_T) of the trap located between the oocyte plasma membrane invaginations and the ISE. The rate at which Na⁺ disappears from the trap during an hyperpolarizing voltage pulse can be estimated (a slope of –1.72 ± 0.30 mV/s at a V_m of –140 mV yields a d[Na⁺]_T/dt of 665 ± 120 µM/s; see Fig. 5). As given in Eq. 2, this d[Na⁺]_T/dt is equal to the change in oocyte Na⁺ flux (J_Na, between –50 and –140 mV) divided by the oocyte geometrical surface (S) x h_T.

(2)

where I_MG is the change in the MG-stimulated current (from –50 to –140 mV), and F is the Faraday constant. Interestingly, the area covered by the ISE cancels out from the calculation because a larger ISE would capture more of the whole oocyte Na⁺ flux but would also dilute these ions in a commensurately larger volume. As shown in Fig. 5, the cotransport current varies from –150 nA at –50 mV to –1,400 ± 100 nA at –140 mV. If we take 0.045 cm² as the geometrical surface area of a typical oocyte (radius 600 µm), h_T can be estimated at 4.9 ± 0.5 µm. As electron micrographs of oocytes show the presence of membrane invaginations in the first 10 µm (1) of the oocyte surface, this estimation of the average trap height appears quite reasonable.

Advantages and limitations of the ion-trap technique. The ion-trap technique is characterized by the combination of a large ISE and the presence of a constricted space where ion concentrations can increase or diminish. This combination allows for the detection of ion transport across membranes without having to rely on transmembrane current measurements or isotope flux measurements. The ion-trap technique has a much better time resolution than isotope flux measurement since changes in transport activities can be detected for an electrogenic transporters within 20 ms of the application of a voltage pulse. Compared with electrophysiology, the ion-trap technique has the advantage that, using steady-state measurements, it can be applied to electroneutral transport mechanisms, and the nature of the transported ion does not have to be determined by ion substitutions since the method is based on the direct detection of a transported ion. Extracellular measurements have previously been done with both static and vibrating probe methods (for examples, see Refs. 6, 14, 16, 17). With respect to vibrating probes, the ion-trap technique provides much better time resolution. With respect to simple static extracellular ISE, the advantage of the ion-trap technique relies on the presence of a microscopic detection volume (the trap), which provides better sensitivity as changes in ion transport will generate a larger change in ionic concentrations within this restricted space vs. the open extracellular space. Although not specifically employed as such in this study, the ion-trap technique could be used to study the distribution of a given transport activity at a different location on the oocyte surface (e.g., difference between animal and vegetal poles). This would probably be used to provide a qualitative description as the seal resistance and the trap volume are expected to change each time a given ISE is put in contact with the oocyte membrane.

One of the most serious disadvantages of the ion-trap technique is that the amplitude of the ion transport activity can only be determined relative to the transport measured in given basal condition. Since the ISE is pushed against the surface of an oocyte at resting potential, the ion flux through the membrane patch covered by the ISE is exactly balanced by the flux across the seal resistance between the ISE and the oocyte membrane. If a substrate is added, or if the V_m is changed, the ion-trap technique will only yield an estimation of the change that has occurred in the membrane ion flux with respect to the starting conditions.

Conclusion. We have presented a method that allows accurate measurement of ionic transport activities without using isotopes or having to measure the transport current. The method is based on the measurement of ion capture and release in a reduced space between an ISE and the oocyte plasma membrane. The method presents good sensitivity and a time resolution that can reach 20 ms. Using the Na⁺/glucose cotransporter to test the ion-trap technique, we were able to demonstrate proportionality between the charge transferred and the surface rapid V_H or V_Na. Moreover, there was direct proportionality between the cotransport currents and the slopes in the ISE signal. On a slower time scale (on the order of 20 s), the activity of the cotransporter can be detected without using its electrogenic properties, simply by adding the substrate. This demonstrates that the ion-trap technique can be applied to electroneutral ion transport as well. We predict that this method will be useful for testing transport mechanisms by looking at a different parameter than the traditional transport current or radioisotope uptake experiments.

	GRANTS

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This work was supported by the Canadian Institutes of Health Research Grant MOP-10580.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J.-Y. Lapointe, Groupe d'étude des protéines membranaires (GÉPROM), Université de Montréal, C.P. 6128, Succ. "Centre-ville," Montréal, Québec, Canada H3C 1J7 (e-mail: jean-yves.lapointe{at}umontreal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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