Name: application of electrophysiology measurement to study the activity of electro-neutral transporters
Uploaded: 2018-02-03T14:00:00.000+00:00
Duration: 11 min 51 s
Description: Watch this Scientific Journal Video about Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters at JoVE.com

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).

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 &#176;C.
1. Preparation of Pipettes for ISEs
<ol>
	<li>Pull the pipettes from borosilicate glass capillaries to an outer diameter of 1.2 mm (see Table of Materials) using the pipette puller2 .</li>
	<li>Polish the pipette using the microforge. The diameter of the pipette tip should be 2-3 &#181;m.</li>
	<li>Place the pipettes in a jar holder for pipettes with the pipette tip facing up (see Table of Materials). 
	CAUTION: Perform steps 1.4-1.6 in a well-ventilated chemical fume hood.</li>
	<li>Mix the siliconization mixture that is composed of 1 drop (~150 &#181;L) of bis(dimethylamino)dimethyl silane and 10 drops of carbon tetrachloride (see Table of Materials).</li>
	<li>Pour the siliconization mixture into the jar and close the lid tightly.</li>
	<li>Incubate the jar with the siliconization mixture for 24-48 h in a chemical fume hood at room temperature until the mixture is completely evaporated.</li>
	<li>Backfill the pipette with the appropriate ion-selective resin to create a column of approximately 2-4 mm. 
	NOTE: Hydrogen ionophore I cocktail B can be usedfor recording NHE activity (see Table of Materials).</li>
	<li>Tap the tip of the pipette to distribute the ion-selective resin to the tip, and place the pipette vertically in the jar holder with the pipette tip down.</li>
	<li>Checkunder the dissecting microscope that the resin cocktail has reached the end of the tip. If needed, apply positive pressure to push the resin to the end.</li>
	<li>Backfill the half-pipette with the appropriate solution (~ 100 &#956;L). Here, use 100 mM KCl and 10 mM HEPES at pH 7.0 if using ionophore I cocktail B.</li>
	<li>Connect the ISE to the electrometer and place the tip of the ISE into the bath solution used for recording. Zero the electrometer.</li>
	<li>Test the H+-selective ISE response by changing the pH of the bath solution. The average H+-selective ISE response is ~ 58 mV per unit of pH.</li>
	<li>Periodically evaluate the ISE response to calibration.</li>
</ol>
2. Preparation of Patch Clamp Pipettes for Recording
NOTE: The method described by Donald Hilgemann in his article on Single-Channel Recording21 is recommended.
<ol>
	<li>Pull the pipettes for giant patch from borosilicate glass capillaries (2.0 mmOD / 1.5 mm ID) on the pipette puller2,22,23.</li>
	<li>Use a microforge mounted on the stage of an inverted microscope to make a wide pipette tip (10 - 22 &#956;m)23. Melt a bead (~ 0.5 mL) of soft glass onto the thick platinum wire of the microforge. 
	NOTE: A relatively thick platinum wire is required (~0.5 mm) to fabricate giant patch pipettes.</li>
	<li>Position the patch pipette close to the soft glass bead, and apply full heat until the tip begins to recede. The platinum wire will move slightly toward the centerduring heating.</li>
	<li>Turn off the heat, and push the pipette tip into the softened glass bead. The cooling wire will retract and break the pipette tip.</li>
	<li>Reapply the heat to smooth the pipette tip and create a pipette tip of the desired diameter. The diameter of the tip is dependent on the size of the cell to be patched (8 - 10 &#956;m in this study).</li>
</ol>
3. Preparation of Cells
<ol>
	<li>Warm up phosphate buffer saline (PBS) without Ca2+ and Mg2+, trypsin-EDTA solution and growth medium to at 37 &#176;C.</li>
	<li>Wash cells twice with PBS. Use 1 mL of the buffer for 35 mm diameter cell culture plate.</li>
	<li>Add 0.5 mL of trypsin to the cells for 35 mm diameter cell culture plate. 
	Note: When cells start to round-up, shake the plate to de-attach the cells.</li>
	<li>Stop the trypsin action by adding 5 mL (amount for 35 mm diameter cell culture plate) of complete growth medium supplemented with 10% fetal bovine serum. 
	NOTE: The reaction time in trypsin is cell-type dependent.</li>
	<li>Place the cells on a shaker with rocking to prevent formation of clumps.</li>
	<li>Use cells for up to 2 h after trypsinization. 
	NOTE: The time might vary depending on the cell line.</li>
</ol>
4. Preparation of an Intra-Pipette Perfusion System
<ol>
	<li>Cut an appropriate size of quartz tubing (150 &#181;m OD / 75 &#181;m ID) to prepare the intra-pipette perfusion line.</li>
	<li>Insert one side of the quartz tubing into a 200 &#181;L pipette tip. Glue the quartz tubing onto the tip, and cut the end of the tip with a sharp razor blade.</li>
	<li>Connect the perfusion line (quartz tubing glued to the tip) to a small piece of silicon tubing that would serve as a reservoir for the intra-pipette perfusion solution.</li>
	<li>Connect the silicon tubing to a syringe to create positive pressure.</li>
	<li>Insert the free end of the quartz tubing through the polyethylene tube of the pipette holder of the perfusion port (see Table of Materials).</li>
	<li>Evaluate the perfusion line with the pipette solution.</li>
</ol>
5. Preparation of Solutions
<ol>
	<li>Prepare a heavily buffered pipette solution for recording NHE (115 mM potassium aspartate (KAsp), 1 mM EGTA, 0.5 mM MgCl2, 10 mM Mg-ATP, 12.5 mM HEPES, 12.5 mM PIPES, 12.5 mM MOPS, and 12.5 mM MES, pH 6.0 adjusted with aspartic acid). 
	NOTE: Prepare the stock pipette solution and filter. Add Mg-ATP before recording.</li>
	<li>Prepare a fresh bath solution each time (140 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 0.1 mM Tris-HCl pH 8.2).</li>
</ol>
6. Recording of Transporter Activity
<ol>
	<li>Maintain the recording chamber and all solutions at 37 &#176;C, because transporter activity is temperature-dependent.</li>
	<li>Place the cells into the recording chamber. Wait until they drop to the bottom of the chamber but do not let them attach to the chamber.</li>
	<li>Choose an appropriate cell. Measure the diameter of the cell with the ocular micrometer. Choose cells with similar diameters. 
	NOTE: Generally, a large cell will have more cellular surface and possibly more transporters expressed on its plasma membrane, thus also having more transporter activity. However, this is not necessarily correct, as transporter activity depends on the specific transporter and the cell type under study.</li>
	<li>Mount the ion-selective microelectrode pipette to the micromanipulator, backfill the patch-clamping pipette with the intracellular solution, make sure that the solution reaches the tip of the pipette2. 
	NOTE: It might require several gentle taps to eliminate bubbles in the tip of the pipette.</li>
	<li>Mount the patch-clamping pipette to the head stage of the electrophysiology set up2,22,23. 
	NOTE: It is important to keep the head stage at about a 45-degree angle to the horizontal axis, as this ensures an entry angle for the pipette that is suitable for the formation of a highly resistant seal.</li>
	<li>Apply a small positive pressure (~ 5 cm H2O) to the patch pipette to reduce the chance of blocking the tip, and place it into the bath solution.</li>
	<li>Stop moving the patch clamp pipette once the tip is close to the cell.</li>
	<li>Relieve the positive pressure while pipette touches the cell and apply slight negative pressure (~ 5 cm H2O) to obtain a &#62;1000 M&#937; (a Giga-ohm) seal on the cell membrane with the patch pipette23.</li>
	<li>Place the patch pipette with the cell in the same focal plane as the ISE, and move the patch pipette with the cell in proximity to the ISE (~ 5 - 10 &#181;m)2,5 but avoid allowing the cell to contact the ISE (Figure 1A).</li>
	<li>Assure that the holding potential is 0 mV.</li>
	<li>Rupture the seal with series of short suctions to obtain the whole-cell configuration.</li>
	<li>Move the ISE (or patch clamp pipette) slowly left-right or up-down to record the activity of the transporter (Figure 1B).</li>
	<li>Record at least 5-8 peaks (Figure 1C, from A to B).</li>
	<li>Apply square wave perturbations during the recording to monitor the quality of the seal and the cell membrane capacitance (Figure 1).</li>
	<li>Estimate the H+ flux (<img alt="Equation 1" src="/files/ftp_upload/56630/56630eq1.jpg" />) from the difference in free H+ concentration between the cell surface and the bulk solution (&#916;H+) 
	<img alt="Equation 2" src="/files/ftp_upload/56630/56630eq2.jpg" /> 
	<img alt="Equation 3" src="/files/ftp_upload/56630/56630eq3v2.jpg" /> 
	NOTE: <img alt="Equation 4" src="/files/ftp_upload/56630/56630eq4.jpg" /> and&#160;<img alt="Equation 5" src="/files/ftp_upload/56630/56630eq5.jpg" /> are H+ and buffer diffusion coefficients respectively, BT is the total buffer concentration, KD is the dissociation constant of the buffer, r is the cell radius, and <img alt="Equation 6" src="/files/ftp_upload/56630/56630eq6.jpg" /> determines the steady-state change in the concentration of bound H+ for a small change in free H+. H+ indicates a free proton in solution. To be consistent with previous studies, H+ fluxes are converted to electrophysiological flux units by calculating the current equivalents of the fluxes (<img alt="Equation 1" src="/files/ftp_upload/56630/56630eq1.jpg" />/faraday) in pA3,5].</li>
	<li>Normalize the proton flux to the cell surface that can be calculated from the cell size or to the membrane capacitance.</li>
</ol>

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The authors have no conflicts of interest to disclose.

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 compositions<...

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 (&#181;V). Voltage differences can be converted into ion flux values using a calibration method that applies Fick&#39;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.

<table><tbody><tr><td>Patch clamp Amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Dual Channel Differential Electrometer (HiZ-223)</td><td>Warner Instruments</td><td>64-1650</td><td></td></tr><tr><td>Differential Amplifier (DP-301)</td><td>Warner Instruments</td><td>64-0044</td><td></td></tr><tr><td>Patch Clamp Software Based on MatLab</td><td>MatLab with acquisition toolbox</td><td></td><td>The capmeter software is recommended</td></tr><tr><td>ThermoClamp-1 Temperature Control System</td><td>&amp;nbsp;AutoMate Scientific</td><td>03-11-LL</td><td>In-line heater</td></tr><tr><td>Single-Channel Temperature Controller (TC-324C)</td><td>Warner Instruments</td><td>64-2400</td><td></td></tr><tr><td>Single-Barrel Standard Borosilicate Glass Tubing</td><td>World Precision Instruments</td><td>1B120F-3&amp;nbsp;&amp;nbsp;</td><td>Used for ion selective electrodes</td></tr><tr><td>Micropipette Storage Jar</td><td>World Precision Instruments</td><td>E212</td><td></td></tr><tr><td>Bis(dimethylamino)dimethylsilane</td><td>Sigma-Aldrich</td><td>14755-100ML</td><td></td></tr><tr><td>Carbon tetrachloride</td><td>Sigma-Aldrich</td><td>319961-500ML</td><td></td></tr><tr><td>Hydrogen ionophore I - cocktail B</td><td>Sigma-Aldrich</td><td>95293</td><td></td></tr><tr><td>Thin Wall Borosilicate Tubing</td><td>&amp;nbsp;Sutter Instrument</td><td>B200-156-15</td><td>Used for patch clamp pipette&amp;nbsp;</td></tr><tr><td>Soft glass (Corning 8161 Patch Glass)</td><td>Warner Instruments</td><td>64-0815</td><td></td></tr><tr><td>Silica Capillary Tubing (150um OD/75um ID)</td><td>Molex (Polymicro Technologies)</td><td>106815-0018</td><td>Used for intra-pipette perfusion system</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Potassium aspartate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A6558</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M4880</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma-Aldrich</td><td>A9187</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M3671</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Apyrase</td><td>Sigma-Aldrich</td><td>A6535</td><td></td></tr><tr><td>Phosphatidylinositol(4,5) bisphosphate diC8</td><td>Echelon Biosciences</td><td>P-4508</td><td></td></tr><tr><td>Phosphatidylinositol 3,4,5-trisphosphate diC8</td><td>Echelon Biosciences</td><td>P-3908</td><td></td></tr></tbody></table>

application of electrophysiology measurement to study the activity of electro-neutral transporters

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.

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 ...

Watch this Scientific Journal Video about Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters at JoVE.com

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

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. ...

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Research

JoVE Journal

Biology

6.9K Views.  Des Moines University. 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.

Video: Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as ...

Biochemistry

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

Recapitulation of an Ion Channel IV Curve Using Frequency Components

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|&lt;0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.

There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into ...

A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of membrane carrier proteins reconstituted in lipid bilayer membranes is a powerful but delicate method for the assessment of their physicochemical and pharmacological properties. The substrate turnover number is a unique parameter that allows the comparison of the activity of different membrane proteins. In an electrogenic transport, the gradient of the translocated substrate creates a membrane potential that directly correlates to the substrate turnover rate of the protein. By using silver chloride electrodes, a diffusion potential, also called liquid junction potential, is induced, which alters electrode potential and significantly disturbs precise membrane potential measurements. Diffusion potential can be minimized by a salt bridge, which balances electrode potential. In this article, a micro-agar salt bridge is designed to improve the electrophysiological set-up, which uses micropipettes for the membrane formation. The salt solution is filled into a microcapillary pipette tip, stabilized by the addition of agarose, and can be easily mounted to a standard electrode. The electrode potential of a micro-salt bridge electrode is more stable compared to a standard electrode. The implementation of this system stabilizes electrode potential and allows more precise measurements of membrane potential generated by a pH gradient. Using this system, the proton turnover rates of the mitochondrial carriers UCP1 and UCP3 are reinvestigated and compared to earlier measurements.

In electrophysiological measurements, the presence of a diffusion potential disturbs the precise measurement of the reverse potential by altering the electrode potential. Using a micro-agar salt bridge, the impact of the diffusion potential is minimized, which allows a more precise measurement of substrate turnover numbers of reconstituted recombinant membrane proteins.

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of ...

Neuroscience

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

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

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

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 ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

Bioengineering

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

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Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Recapitulation of an Ion Channel IV Curve Using Frequency Components

A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

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

Introduction to Solid Supported Membrane Based Electrophysiology

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl^- Channels and Transporters

Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane