Name: application of electrophysiology measurement to study the activity of electro-neutral transporters
Uploaded: 2018-02-03T14:00:00
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...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="961">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Patch clamp Amplifier</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:101px;"></td>
			<td style="width:276px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Dual Channel Differential Electrometer (HiZ-223)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-1650</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Differential Amplifier (DP-301)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-0044</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Patch Clamp Software Based on MatLab</td>
			<td style="width:219px;">MatLab with acquisition toolbox</td>
			<td style="width:101px;"></td>
			<td>The capmeter software is recommended</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">ThermoClamp-1 Temperature Control System</td>
			<td>&#160;AutoMate Scientific</td>
			<td style="width:101px;">03-11-LL</td>
			<td>In-line heater</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Single-Channel Temperature Controller (TC-324C)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-2400</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Single-Barrel Standard Borosilicate Glass Tubing</td>
			<td>World Precision Instruments</td>
			<td style="width:101px;">1B120F-3&#160;&#160;</td>
			<td style="width:276px;">Used for ion selective electrodes</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Micropipette Storage Jar</td>
			<td>World Precision Instruments</td>
			<td>E212</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Bis(dimethylamino)dimethylsilane</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>14755-100ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Carbon tetrachloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td colspan="2">319961-500ML</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Hydrogen ionophore I - cocktail B</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">95293</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Thin Wall Borosilicate Tubing</td>
			<td style="width:219px;">&#160;Sutter Instrument</td>
			<td style="width:101px;">B200-156-15</td>
			<td style="width:276px;">Used for patch clamp pipette&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Soft glass (Corning 8161 Patch Glass)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-0815</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Silica Capillary Tubing (150um OD/75um ID)</td>
			<td style="width:219px;">Molex (Polymicro Technologies)</td>
			<td>106815-0018</td>
			<td style="width:276px;">Used for intra-pipette perfusion system</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Potassium chloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Potassium aspartate&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>A6558</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">EGTA</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Magnesium chloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">M4880</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Mg-ATP</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9187</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">HEPES</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">PIPES</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">MOPS</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">MES</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>M3671</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Calcium chloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Tris</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">T1503</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Hydrochloric acid</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Apyrase</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>A6535</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Phosphatidylinositol(4,5) bisphosphate diC8</td>
			<td>Echelon Biosciences</td>
			<td>P-4508</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">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 ...

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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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