This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.
The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.
Transport of ions and solutes across the plasma membrane is essential for the survival of cells and, hence, of organisms1. Selective transport of ions and solutes is achieved by an array of specialized channel and transporter proteins. Mutations in these proteins often result in a variety of clinical conditions, rendering channel and transporter proteins potential targets for pharmacological treatment1. Unfortunately, understanding the mechanisms underlying channel and transporter function and regulation is often limited by the approaches available to study their activity2,3,4.
Specifically, transporters can be roughly sub-divided into two large groups depending on whether they alter the cell transmembrane potential during transport of solutes: the altering electrogenic ion transporters [e.g., sodium-phosphate co-transporter 2a (NaPi2a), sodium-calcium exchanger (NCX), etc.] or the non-altering electroneutral ion transporters [e.g., sodium-proton exchanger (NHE), sodium-chloride co-transporter, NaPi2c, etc.]. The activities of both classes of transporters have been studied extensively using uptake of radioactive isotopes and fluorescent membrane-permeant dyes2. Both approaches estimate the activity of transporters by measuring changes in the bulk concentration of specific cytoplasmic ions, and both methods have limitations, such as moderate sensitivity and time resolution and inadequate control of the intracellular milieu. Indeed, the activity of many transporters is dependent on the cytoplasmic concentration of the carried ions (e.g., NHE3, NCX), and changes in these ion concentrations are expected to play a significant role in regulating transporter activity2,3,5. Precise measurement of these regulative mechanisms is limited using classical methods.
To overcome these limitations, patch clamp methods are used to study the transporter activity2,6. Specifically, the self-referencing ion-selective electrodes (ISEs)7,8 combined with the patch clamping system has recently allowed the measurement of electroneutral transporter activity3,4,5. ISEs are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An ISE moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference (µV). Voltage differences can be converted into ion flux values using a calibration method that applies Fick's first law of diffusion2,9. While ISEs are used to detect the flux of ions moving out of cells, the patch clamp method in both whole-cell or inside-out configurations is used to control the membrane potential and intracellular ion composition. Moreover, the application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids and proteins3,5.
In summary, the versatility of the patch clamp method compared with that of other methods to study transporter activity has made patch clamping suitable to overcome the common limitations of these other methods. The combination of self-referencing ISEs and patch clamp techniques offers the unique possibility to measure the activity of electroneutral transporters in a tightly controlled experimental environment and to discover novel biophysical and molecular properties of cell membrane transport3,4,5. This approach has been successfully used to study the activity of the NHE. The mammalian NHE protein family catalyzes the electroneutral net exchange of extracellular sodium (Na+) for intracellular proton (H+)10,11 utilizing an inward Na+ gradient. In mammals, the NHE protein family includes 11 related proteins (NHE1-9 and NHA1-2) and a sperm-specific NHE10,12,13.
NHEs (SLC9a family) are found ubiquitously in most living organisms from simple prokaryotes to higher eukaryotes and are involved in a variety of vital cell functions10,11, including controlling the cell salinity defense in prokaryotes, maintaining acid-base homeostasis and cell volume, and regulating the absorption of salt and water in various specialized epithelia10,12,14,15. The key biological roles of NHEs and the significance of their functions have been determined through several studies; however, few studies have investigated the biophysical and molecular properties of mammalian NHEs because of methodological limitations4. Recently, the application of self-referencing ISEs during whole-cell patch clamping has revealed novel molecular mechanisms of NHE isoforms regulated by changes in the intracellular concentrations of ions, proteins and phospholipids3,4.
Specifically, the protocol provided in this manuscript outlines the methods and approaches for studying the activity and regulation of NHE isoform 3 (NHE3), a major player in the absorption of Na+, Cl–, HCO3– and fluid in the brush border membrane of renal and intestinal epithelia14,16. New insight into differences in the sensitivity of NHE3 activity to intracellular phosphoinositides (phosphatidylinositide 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositide 3,4,5-triphosphate [PI(3,4,5]P3]) is reported. Cell transport proteins, such as channels and transporters, are regulated by phosphoinositides17, and NHE3 directly binds both PI(4,5)P2 and PI(3,4,5)P318. Based on the current literature, either phosphoinositide could be relevant for the physiological or pathophysiological regulation of NHE35,18,19. Our findings support separate roles for PI(4,5)P2 and PI(3,4,5)P3 in the regulation of NHE3 activity. This distinction was possible because of the application of ISE techniques in combination with whole-cell patch clamp recording. This technique also allows control of the phosphoinositide cellular content via the intracellular perfusion of different phosphoinositides during the measurement of NHE3 activity.
Note: Two amplifiers are required for recording the activity of electroneutral transporters by a self-referencing ISE in conjunction with patch clamping, a patch clamp amplifier to maintain the cell in a whole-cell configuration and a high impendence amplifier (electrometer) to record the transporter activity via the ISE (see Table of Materials). The patch clamp amplifier is directly connected to the acquisition board terminal. The electrometer is connected to the differential amplifier utilized to filter and amplify the signal. The differential amplifier is connected to the acquisition board terminal. Capmeter, patch clamp software that monitors cell capacity, has been used in previous studies20 (see Table of Materials). The recording chamber and perfusion solutions are maintained at 37 °C.
1. Preparation of Pipettes for ISEs
2. Preparation of Patch Clamp Pipettes for Recording
NOTE: The method described by Donald Hilgemann in his article on Single-Channel Recording21 is recommended.
3. Preparation of Cells
4. Preparation of an Intra-Pipette Perfusion System
5. Preparation of Solutions
6. Recording of Transporter Activity
A self-referencing ISE during whole-cell patch clamp recording was applied to study the regulation of NHE3 activity by phosphoinositides. PS120 fibroblast-like cells24, which lack the expression of endogenous plasma membrane NHEs, were used. NHE3 wild-type (NHE3-wt) or NHE3 mutants that do not bind phosphoinositides [tyrosine 501, arginine 503 and lysine 505 were substituted with alanine, Y501A/R503A/K505A (NHE3-YRK)] were stably expressed in PS120 fibroblast-like cells18.
The traces are presented in Figure 2. The cell was heldin a whole-cell configuration by a pipette for patch clamping, and the ISE was placed in front of the cell. In this position, the ISE recorded the concentration of free H+ close to the cell membrane (Figure 1A); then, the ISE (or patch clamp pipette) was manually moved laterally 50 µm and recorded the concentration of free H+ in the solution (Figure 1B). The recorded voltage differences, corresponding to the two positions of the patch pipette with the cell in front of or far from the ISE, are representative of NHE3 activity (Figure 1C). The intra-pipette perfusion of apyrase (ATP-diphosphatase) decreases the intracellular concentration of ATP, subsequently decreasing the phosphoinositide content5. In agreement with previously reported studies5,18,19, the decreased phosphoinositide content mediated by apyrase treatment reduced NHE3 activity (~50%), which was recorded as a decrease in the voltage oscillation amplitude (Figure 2). The effect of apyrase (given at 5 units/ml) on NHE3 activity was completely reversed by the intra-pipette perfusion of apyrase in combination with 2 µM PI(3,4,5)P3 (Figure 2 and 3A). These findings were supported by the NHE3 activity recorded in PS120 cells stably expressing NHE3 mutants (NHE3-YRK) that were unable to bind phosphatidylinositides18. The perfusion of apyrase alone or in combination with PIP3 did not affect the activity of NHE3 in NHE3-YRK mutants (Figure 3B). The activity of endogenous NHE3 was also inhibited by apyrase treatment in opossum kidney (OK) cells3. The intra-pipette perfusion of PI(3,4,5)P3 reversed the inhibition of NHE3 activity induced by apyrase in OK cells (Figure 3C). These findings suggest that the effect of PI(3,4,5)P3 on NHE3 activity is not cell type-specific.
Next, we aimed to test the specificity of PIP3 on regulating apyrase-mediated changes in NHE3 activity. We did this by assaying the effect on apyrase-dependent reduction on NHE3 activity after the perfusion of other phosphoinositides, such as PI(4,5)P2. PI(4,5)P2 (at 10 µM) did not affect the decreased NHE3 activity mediated by apyrase (Figure 4A). Finally, the perfusion of AMP-PNP, a non-hydrolysable ATP analog, did not block theapyrase-dependent inhibition of NHE3 activity (Figure 4B). These findings suggest a role of PI(3,4,5)P3, but not PI(4,5)P2, in the regulation of NHE3 after ATP depletion. The identification of separate roles for PI(4,5)P2 and PI(3,4,5)P3 in NHE3 regulation was possible only because of the intercellular perfusion of phosphoinositides during ISE measurements combined with whole-cell patch clamp recording5. A specific action of PI(3,4,5)P3 rather than PI(4,5)P2 in the regulation of NHE3 activity is valuable to determine whether different intracellular phosphoinositides regulate the function of NHE3 differently.
Figure 1. Schematic representation of H+ flux measurement and correlation between NHE activity and potential differences recorded by the pH microelectrode.
Schematic representation of the recording setting. A. Diagram of the initial experimental setting. The cell is held in the whole-cell configuration by the patch clamp pipette and placed in front of the ISE. In this position, the ISE records the concentration of free H+ close to the cell membrane. B. The ISE (or patch clamp pipette) is manually moved laterally ~50 µm and records the concentration of free H+ in the solution in this position. C. Graphic representation of voltage differences recorded using the ISE. The oscillations from position A (cell in front of the ISE) to B (cell far from the ISE) are representative of NHE activity. The software used registered the voltage differences recorded by the ISE and generated square wave perturbations (20 mV, 0.2 kHz) to monitor the cell membrane capacitance and quality of the patch clamp seal. Please click here to view a larger version of this figure.
Figure 2. Example of a recording set for NHE3 transporter activity in a single PS120 cell stably expressing NHE3-wt before and after perfusion with apyrase and PI(3,4,5)P3 (PIP3).
After the initial recording, the cell was depleted of ATP by the intra-pipette perfusion of apyrase. NHE3 activity gradually decreased and was restored by PIP3 intra-pipette perfusion. Apyrase addition is indicated by the red bar, and PIP3 addition is indicated by the green bar. Please click here to view a larger version of this figure.
Figure 3. Effect of ATP depletion and PIP3 perfusion on NHE3 transporter activity in PS120 and OK cells.
Effect of apyrase-mediated ATP depletion on NHE3 activity and reserving the effect of apyrase on NHE3 activity induced by PIP3 intra-pipette perfusion: A. PS120 cells expressing NHE3-wt or B. PS120 cells expressing NHE3-YRK, NHE3 mutants unable to bind phosphoinositides. C. OK cells expressing endogenous NHE3. Solid circles show average data from individual experiments (four experiments performed in identical conditions). Error bars represent the means ± standard errors (SE). *P < 0.05 vs. control, ANOVA. $P< 0.5, $$P < 0.01 apyrase vs. apyrase + PIP3, ANOVA. Please click here to view a larger version of this figure.
Figure 4. Effect of PI(4,5)P2 (PIP2) and ANP-PNP perfusion on NHE3 activity after ATP depletion.
Effect of A. PIP2 or B. ANP-PNP intra-pipette perfusion on the apyrase-dependent reduction of NHE3 activity in PS120 cells expressing NHE3-wt. Solid circles show average data from individual experiments (four experiments performed in identical conditions). Error bars represent the means ± SE. *P < 0.05, **P < 0.01 vs. control, ANOVA. Please click here to view a larger version of this figure.
Despite the crucial functions of transporters, the methods available to study their activity are ineffective and inadequate. One limitation is that the available methods measure ion movement mediated by transporter activity without considering fluctuations in intracellular ion composition during the experiment4. The presented method ensures precise control of extracellular and intracellular ion compositions and offers a powerful tool for modifying intracellular ion, protein and lipid compositions3,4,5.
The most important step in this protocol is the preparation of an ISE that is stable for a long period25. In our experience, an unstable ISE is not well-siliconized (e.g., the ion-selective resin is redistributed on the ISE pipette sides), which can be detected by a constant and uncontrollable drift in the ISE signal. Additionally, the resin distribution in the ISE should be closely monitored during the experiment. The bath solution may push the ion-selective resin inside the ISE pipette, creating a column of bath solution before the resin25. In this case, the ISE will record the ion concentration from this column and become nonresponsive to changes in the ion gradients during the experiment. Adjusting the geometry of the ISE is key to obtain an ISE response in the range of 100 ms9 and overcome possible delays in ISE time-responses compared with changes in ion gradients.
The major limitation of the presented method is inherited from the patch clamping measurement of transporter activity in epithelial cells. When detached from the support and maintained in suspension, polarized epithelial cells might lose the polarized localization of transporters on the apical or basolateral membrane.
A modified patch clamping measurement should be used when studying channels and transporters in polarized epithelia. Considering that cells might approximate into a cylinder rather than a sphere is important when transforming the H+ gradient into flux via the formula provided. In this case, the formula should be changed to where λ is the length of the cylinder, r is the cylinder radius, ad x is the radial distance from the cylinder midpoint over which the gradient is measured5. Future development of this powerful technique for studying electroneutral transporters should focus on modifying the method to optimize recording cells grown in a monolayer.
The authors have nothing to disclose.
The authors would like to thank Eric Fishback (Des Moines University, Des Moines, Iowa, USA) for his assistance with shooting and editing the video. The PS120 fibroblast-like cells stably expressing NHE3-wt or NHE3-YRK were kindly provided by Dr. Mark Donowitz (Johns Hopkins University School of Medicine, Baltimore, MD, USA).
Patch clamp Amplifier | Molecular Devices | ||
Dual Channel Differential Electrometer (HiZ-223) | Warner Instruments | 64-1650 | |
Differential Amplifier (DP-301) | Warner Instruments | 64-0044 | |
Patch Clamp Software Based on MatLab | MatLab with acquisition toolbox | The capmeter software is recommended | |
ThermoClamp-1 Temperature Control System | AutoMate Scientific | 03-11-LL | In-line heater |
Single-Channel Temperature Controller (TC-324C) | Warner Instruments | 64-2400 | |
Single-Barrel Standard Borosilicate Glass Tubing | World Precision Instruments | 1B120F-3 | Used for ion selective electrodes |
Micropipette Storage Jar | World Precision Instruments | E212 | |
Bis(dimethylamino)dimethylsilane | Sigma-Aldrich | 14755-100ML | |
Carbon tetrachloride | Sigma-Aldrich | 319961-500ML | |
Hydrogen ionophore I – cocktail B | Sigma-Aldrich | 95293 | |
Thin Wall Borosilicate Tubing | Sutter Instrument | B200-156-15 | Used for patch clamp pipette |
Soft glass (Corning 8161 Patch Glass) | Warner Instruments | 64-0815 | |
Silica Capillary Tubing (150um OD/75um ID) | Molex (Polymicro Technologies) | 106815-0018 | Used for intra-pipette perfusion system |
Potassium chloride | Sigma-Aldrich | P9333 | |
Potassium aspartate | Sigma-Aldrich | A6558 | |
EGTA | Sigma-Aldrich | E3889 | |
Magnesium chloride | Sigma-Aldrich | M4880 | |
Mg-ATP | Sigma-Aldrich | A9187 | |
HEPES | Sigma-Aldrich | H3375 | |
PIPES | Sigma-Aldrich | P6757 | |
MOPS | Sigma-Aldrich | M1254 | |
MES | Sigma-Aldrich | M3671 | |
Calcium chloride | Sigma-Aldrich | C5670 | |
Tris | Sigma-Aldrich | T1503 | |
Hydrochloric acid | Sigma-Aldrich | H1758 | |
Apyrase | Sigma-Aldrich | A6535 | |
Phosphatidylinositol(4,5) bisphosphate diC8 | Echelon Biosciences | P-4508 | |
Phosphatidylinositol 3,4,5-trisphosphate diC8 | Echelon Biosciences | P-3908 |