Name: Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes (Video) | JoVE Journal
Uploaded: 2010-12-21T15:00:00.000+00:00
Duration: 15 min 29 s
Description: Learn whole-cell patch clamp recording of CRAC currents in human T lymphocytes using peripheral blood mononuclear cells. This reliable method enables precise electrophysiological measurements for studying calcium signaling in immune cell activation.

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5 minutes

We are thankful to the Department of Physiology and Membrane Biology, University of California Davis for providing us with facilities and an excellent environment for the studies of ion channels.

1. Preparation of Resting Human T Lymphocytes
<ol>
 <li>Using RosetteSep Human T Cell Enrichment Cocktail and RosetteSep Density Medium, purify T cells from human blood samples according to the manufacturer's instructions. The resulting cell population should contain 95% CD3+ resting T cells. We purify human T lymphocytes from peripheral blood samples collected from healthy volunteers in accordance with the protocol approved by the UC Davis Internal Review Board.</li>
 <li>After isolation, resuspend resting T cells at a density of 0.5 x 106 cells/ mL in cell culture medium containing RPMI-1640 medium with glutamine and HEPES, supplemented with 10% fetal calf serum, 2% GlutaMAX-I, 1% RPMI 1640 vitamin solution, 1% RPMI 1640 amino acids solution, 1% sodium pyruvate, and 0.03% β-mercaptoethanol.</li>
 <li>Place cell suspension into cell culture flasks and maintain at 37°C in a humidified incubator with 5% CO2 for up to 24 h prior to the experiment.</li>
</ol>
2. Preparation of the Recording Chamber
<ol>
 <li>The following are needed to prepare recording chambers: a) silicon O-rings, b) round, 25-mm coverslips cleaned with 70% ethanol and distilled H2O, c) mixed Sylgard184, prepared according to the manufacturer's instructions, and d) two 60 x 15 mm Petri dishes.</li>
 <li>Place ~0.5 mL of mixed Sylgard184 into a 60x15-mm Petri dish. </li>
 <li>Dip the bottom part of the O-ring into the Sylgard184 using forceps.</li>
 <li>Remove excess Sylgard184 from the rim of the O-ring by placing the O-ring on a clean surface, such as another Petri dish. </li>
 <li>Place the ring onto the coverslip and press it down.</li>
 <li>Cure the Sylgard184 by heating the chambers at 85° C for 40-60 min or until the Sylgard184 is polymerized.</li>
 <li>Store the recording chambers in a dust-free container.</li>
</ol>
3. Preparation of Patch Pipettes
<ol>
 <li>On the day of the experiment, pull pipettes with a ~2-μm tip diameter using glass capillaries and a pipette puller. We use borosilicate tubing with filament (O.D.: 1.5 mm and I.D.: 1.10 mm). Store pipettes in a dust-free location.</li>
 <li>Coat pipette tips with Sylgard184 or HIPEC R6101 Semiconductor Protective Coating, according to a standard procedure 9.</li>
 <li>Gently fire-polish the pipettes before the experiment. </li>
</ol>
4. Patch Clamp Setup Preparation
<ol>
 <li>Configure and save the macros for gigaseal formation in the software controlling your patch-clamp amplifier. We use an EPC-10 amplifier supported by Windows XP and Pulse software for the data acquisition. We set the "set-up", "on-cell", and "whole-cell" macros according to the EPC-10 manual and use them for all experiments.</li>
 <li>Prepare bath solutions and pipette solutions listed in Table 1 prior to the experiment and store them at 4° C for up to 1 week.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>Chemicals</td>
 <td colspan="3">Bath Solutions (mM)</td>
 <td>Pipette Solutions (mM)</td>
 </tr>
 <tr>
 <td> </td>
 <td># 1</td>
 <td># 2</td>
 <td># 3</td>
 <td> </td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>CH3SO3Na</td>
 <td>130</td>
 <td>110</td>
 <td>125</td>
 <td>-</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>2</td>
 <td>4</td>
 <td>5</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Ca(OH)2</td>
 <td>-</td>
 <td>20</td>
 <td>-</td>
 <td>-</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>3</td>
 <td>1</td>
 <td>-</td>
 <td>5</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>-</td>
 <td>-</td>
 <td>-</td>
 <td>2</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>15</td>
 </tr>
 <tr>
 <td>HEDTA</td>
 <td>-</td>
 <td> </td>
 <td>10</td>
 <td>-</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>-</td>
 <td>-</td>
 <td>1</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Cs-aspartate</td>
 <td>-</td>
 <td>-</td>
 <td>-</td>
 <td>125</td>
 </tr>
 <tr>
 <td>BAPTA</td>
 <td>-</td>
 <td>-</td>
 <td>-</td>
 <td>12</td>
 </tr>
<tr>
<td>Glucose</td>
<td>10</td>
<td>10</td>
<td>10</td>
<td>-</td>
</tr>
 </tbody>
</table>
Table 1. Solutions for whole-cell CARC current recordings.
All chemicals were purchased from Sigma-Aldrich, St. Louis, MO.
<ol start="3">
 <li>Determine liquid junction potentials (LJ) between the patch pipette solution and each bath solution 10. Save the LJ value in the appropriate place using your acquisition software. For example, in the Pulse program insert the LJ value into "LJ" control in the "Amplifier" window.</li>
 <li>Configure and save the stimulation protocols, as shown in Figure 1, in the appropriate place of your acquisition software before the experiments. In the Pulse program, we set the acquisition protocol in the "Pulse Generation" window. <img src="/files/ftp_upload/2346/2364fig1.jpg" alt="Voltage-clamp method diagram showing membrane potential steps at -120 mV, 30 mV, and 100 mV." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Stimulation protocol. Voltage ramp from -120 to +100 mV of 50 ms in duration is applied every 0.2 - 2 s (5 - 0.5 Hz). Holding potential (Vh) between ramps is maintained at +30 mV.</li>
</ol>
5. Mounting Cells on the Microscope Stage
<ol>
 <li>Coat the recording chambers with poly-L-lysine for ~20 min and wash with H2O and then with Hank's 	balanced salt solution (HBSS) before plating the cells.</li>
<li>Plate 	500 μL of cell suspension containing 0.2-0.5 x106 cells into the chamber and incubate for 20 min at 37° C.</li>
<li>Secure 	the recording chamber with the attached cells in a chamber holder. 	We use a custom-made chamber holder, where the base supports the 	bottom of the coverslip, while the top part gently presses against 	the top of the coverslip. </li>
<li>Place 	the chamber holder on the stage of the inverted microscope.</li>
<li>Focus on the cells in transmitted light. We use a 40X oil immersion objective.</li>
<li>Place 	the Ag/AgCl reference electrode into the bath.</li>
<li>Align 	the multibarrel, gravity-driven perfusion system. The tip of the 	inflow tube should be placed at a 45° angle to the coverslip close to the field of view.</li>
<li>Position 	the suction tube opposite to the tip of the inflow tube.</li>
<li>Perfuse 	the chamber with phosphate-buffered saline (PBS) or HBSS to wash 	away the cell culture media and loosely attached cells. </li>
</ol>
6. CRAC Channel Currents Recording
<ol>
 <li>Find a cell that is firmly attached to the coverslip. We typically select an average-sized, round cell with a smooth outer surface. </li>
 <li>Perfuse the recording chamber with Ca2+-free, 3 mM Mg2+-containing bath solution #1 (Table 1) containing 0.5-1 μM thapsigargin for 8-10 min to block the SERCA pump. We perform this step to deplete the store prior to gigaseal formation. </li>
 <li>Using an Eppendorf microloader and a P-20 displacement pipette, fill the patch pipette with the pipette solution (Table 1) and insert the patch pipette into the pipette holder on the amplifier headstage. The Ca2+ chelator BAPTA is included in the pipette solution to passively deplete the store and to reduce Ca2+-dependent CRAC channel inactivation.</li>
 <li>Apply a small amount of positive pressure (~3 inches of H2O) inside the patch pipette before entering the bath. We use a custom-made pressure-suction device connected to pressurized air and vacuum lines to create positive or negative pressure inside a patch pipette. </li>
 <li>Lower the patch pipette into the bath under visual control via the microscope. In the "Oscilloscope" window, you should see a current flowing through the pipette. A step-like change in the current amplitude should be induced by a small amplitude voltage pulse (test pulse) applied by the preset macro. At this time, you should be able to determine the value of pipette resistance. The resistance of our pipettes is typically 5-6 MΩ.</li>
 <li>Correct the "offset" potential generated between the pipette and the reference electrode.</li>
 <li>Under visual control via the microscope, bring the pipette tip closer to the cell until it touches the cell membrane.</li>
 <li>Apply negative pressure (20-40 inches of H2O) inside the patch pipette. </li>
 <li>Monitor the gigaseal formation in the "oscilloscope" window. You will see the amplitude of the current, induced by the test pulse, decreases and the value of pipette resistance increases to &gt;5 GΩ. Usually gigaseal forms within a few seconds, but it may take 1-2 min to achieve a stable gigaseal.</li>
 <li>Compensate for the pipette capacitance ("C-Fast"). </li>
 <li>Break the patch membrane by applying additional negative pressure using a 20 cc syringe.</li>
 <li>Set the holding potential to +30 mV. A positive holding potential helps to prevent calcium-dependent CRAC channel inactivation.</li>
 <li>Compensate for the membrane capacitance of the cell ("C-slow"); save or record the value of C-slow. </li>
 <li>Check the value of the access resistance "Rs". In our experiments, the Rs is typically 7-20 MΩ.</li>
 <li>Start to apply a series of voltage ramps with a frequency of 0.5 Hz (Figure 1) in Ca2+-free 3 mM Mg2+-containing bath solution #1. In this solution, CRAC current is negligibly small as compared with the "leak" current due to the poor permeability of CRAC channels for Mg2+. Save and use the current traces recorded in bath solution #1 in future analyses as "leak" currents. The absolute amplitude of the "leak" current at -100 mV should be less than or equal to 5 pA. If the cell is "leaky", stop the experiment and start over using a new patch pipette and a new cell.</li>
 <li>To record Ca2+ current via CRAC channels (ICa-CRAC), replace Ca2+-free 3 mM Mg2+-containing bath solution #1 with 20 mM Ca2+-containing bath solution #2 (Table 1) by switching the perfusion valves. This allows for Ca2+ to flow via CRAC channels and produce an inward current. In the "Oscilloscope" window, you will see development of inwardly rectifying ICa-CRAC (Figure 2). The current amplitude at negative voltages will continue to increase for about 1 min due to the Ca2+-dependent potentiation of the CRAC current.</li>
 <li>Increase the frequency of stimulation with voltage ramps to 5 Hz, which is necessary to record the transient Na+ current via CRAC channels.</li>
 <li>Replace the 20 mM Ca2+-containing bath solution #2 with Na+-containing divalent cation-free solution #3 (Table 1) by switching the perfusion valves. In the "Oscilloscope" window, you will see a transient development of larger amplitude inwardly rectifying Na+ current via the CRAC channels (INa-CRAC; Figure 2).</li>
 <li>One may apply 20 mM Ca2+-containing bath solution #2 containing 1-5 μM La3+ at the end of the experiment to record the "leak" current. However, we did not find this approach useful because the "leak" current changes over time and it may become larger or smaller at the end of the experiment. We prefer to take the current recorded in the Ca2+-free 3 mM Mg2+-containing bath solution # 1 at the beginning of the experiment as a "leak" current.</li>
 <li>Save the recorded current traces for further analysis.</li>
</ol>
7. Data Analysis
<ol>
 <li>Retrieve the saved current records into analysis software. We use the Pulse program for analysis.</li>
 <li>Measure the current amplitudes at the beginning and at the end of voltage ramps. We typically measure the current amplitudes at -100 mV and at +100 mV using pairs of cursors in the "Oscilloscope" window of the Pulse program. </li>
 <li>Export the values of current amplitudes into a graphic program for further analysis and graphical presentation. We use Origin Scientific Graphing and Analysis Software, version 7.</li>
</ol>
8. Representative Results
<img src="/files/ftp_upload/2346/2346fig2.jpg" alt="Electrophysiology experiment diagrams showing ion current (I) vs. membrane voltage (Vm) analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. CRAC currents in a resting human T cell. (A), Time course of CRAC currents recorded in whole-cell voltage-clamp configuration at -100 mV (filled circles) and +100 mV (open circles). Prior to gigaseal formation, the cell was preincubated for ~10 min in Ca2+-free 3 mM Mg2+-containing bath solution #1 containing 0.5 μM thapsigargin. After break-in, bath solutions were sequentially applied as follows: Ca2+-free 3 mM Mg2+-containing bath solution #1 (0 Ca + 3 Mg), followed by 20 mM Ca2+-containing bath solution #2 (20 Ca), followed by divalent cation-free bath solution #3 (DVF), followed by bath solution #2 (20 Ca). The cell was stimulated with a series of voltage ramps as shown in Figure 1. The frequency of ramps was 5 Hz in bath solution #3 (DVF) and 0.5 Hz in all other solutions. Note the slow development of ICa-CRAC after application of 20 mM Ca2+-containing bath solution #2 and the fast transient development of INa-CRAC in DVF bath solution #3. (B), Representative current traces recorded during voltage ramps in Ca2+-free 3 mM Mg2+-containing bath solution #1 ("leak"), 20 mM Ca2+-containing bath solution #2 (20 Ca), and bath solution #3 (DVF). (C, D), Current-voltage relationships of ICa-CRAC (C) and INa-CRAC (D) obtained by subtracting "leak" current from the currents recorded during voltage-ramps in 20 mM Ca2+-containing bath solution #2 (20 Ca), and bath solution #3 (DVF) shown in panel (B).

<ol><li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Store-operated+CRAC+channels%3A+function+in+health+and+disease%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li>
<li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRAC+channelopathies%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li>
<li>Lewis, R. S., Cahalan, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitogen-induced+oscillations+of+cytosolic+Ca2%2B+and+transmembrane+Ca2%2B+current+in+human+leukemic+T+cells%5BTitle%5D)+AND+%22Cell+Regul%22%5BJournal%5D)">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li>
<li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+inactivation+of+depletion-activated+calcium+current+%28ICRAC%29+due+to+local+calcium+feedback%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li>
<li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li>
<li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li>
<li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2%2B-inhibited+cation+%28MIC%29+channels%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li>
<li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+channel+properties+and+regulated+expression+of+Ca%282%2B%29+release-activated+Ca%282%2B%29+%28CRAC%29+channels+in+human+T+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li>
<li>Sakmann, B., Neher, E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSakmann%2C+B+%22Single-Channel+Recording%22">Single-Channel Recording</a>. , 2nd edition, Plenum Press. New York. (1995).</li>
<li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correction+for+liquid+junction+potentials+in+patch+clamp+experiments%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li>
</ol>

No conflicts of interest declared.

The electrophysiological investigation of CRAC currents in resting human T cells is a challenging task because the endogenous CRAC current amplitude in these cells is small due to the small cell size (the resting human T cell diameter is in the range of 5-8 &mu;m). Here, we present a step-by-step procedure to reliably record CRAC currents in resting human T lymphocytes isolated from peripheral blood mononuclear cells. This technology allows us to investigate the physiology and functional expression of CRAC channels in re...

<table><tbody><tr><td>RosetteSep Human T Cell Enrichment Cocktail</td><td>Stem Cell Technologies</td><td>15061</td><td></td></tr><tr><td>RosetteSep Density Medium</td><td>Stem Cell Technologies</td><td>15705</td><td></td></tr><tr><td>RPMI-in 1640 medium w/glutamine/HEPES</td><td>Fisher Scientific</td><td>SH3025501</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Omega Scientific</td><td>FB-01</td><td></td></tr><tr><td>GlutaMAX-I (100X solution)</td><td>Invitrogen</td><td>35050</td><td></td></tr><tr><td>RPMI 1640 vitamin solution (100X)</td><td>Sigma-Aldrich</td><td>7256</td><td></td></tr><tr><td>1640 amino acids solution (50X)</td><td>Sigma-Aldrich</td><td>R7131</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>&amp;#946;-Mercapt&amp;#8211;thanol</td><td>Sigma-Aldrich</td><td>M7522</td><td></td></tr><tr><td>Inositol trisphosphate</td><td>Sigma-Aldrich</td><td>19766</td><td></td></tr><tr><td>BAPTA</td><td>Sigma-Aldrich</td><td>A4926</td><td></td></tr><tr><td>Poly-L-Lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P2636</td><td></td></tr><tr><td>Lanthanum Chloride</td><td>Sigma-Aldrich</td><td>262072</td><td></td></tr><tr><td>Thapsigargin</td><td>Calbiochem</td><td>586005</td><td></td></tr><tr><td>Sylgard 184 Silicon Elastomer Kit</td><td>Dow Corning</td><td>3097358-1004</td><td></td></tr><tr><td>HIPEC R6101 Semiconductor Protective Coating</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>63-500 Series High-Performance Vibration Isolation Lab Table</td><td>Technical Manufacturing Corp.</td><td>63-540</td><td></td></tr><tr><td>EPC 10 patch clamp amplifier with headstage</td><td>HEKA Instruments</td><td></td><td></td></tr><tr><td>Micromanipulator </td><td>Sutter Instrument Co.</td><td>MP-285</td><td></td></tr><tr><td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td><td>Olympus Corporation</td><td>1X71</td><td></td></tr><tr><td>Windows Computer</td><td>Dell</td><td></td><td></td></tr><tr><td>Pulse software</td><td>HEKA Instruments</td><td></td><td></td></tr><tr><td>Origin Scientific Graphing and Analysis Software</td><td>OriginLab</td><td></td><td></td></tr><tr><td>Patch pipette puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td><td>Sutter Instrument Co.</td><td>BF150-110-7.5</td><td></td></tr><tr><td>Narashige&amp;rsquo;s Microforge</td><td>Tritech Research, Inc.</td><td>MF-830</td><td></td></tr><tr><td>Silicon O-rings </td><td>McMaster-Carr</td><td>111 S70</td><td></td></tr><tr><td>Coverslips 25 mm </td><td>Fisher Scientific</td><td>12-545-102 25 mm 25CIR.-1</td><td></td></tr></tbody></table>

whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Calcium release activated calcium channels or crack channels, which are located in the plasma membrane play an important role in T-cell proliferation and gene expression stimulation of T-cell receptors with an antigen results in formation of the second messenger, anatol tri phosphate and activation of calcium release from the store via anatol RIF phosphate receptors. Reduction in calcium concentration within the store leads to activation of crack channels and consequent increase in the cytosol calcium concentration. Abnormalities in crack channel function in T cells are linked to a number of human diseases, including severe combined immunodeficiency disorders and autoimmune diseases.Here a method is presented that allows for reliable and reproducible recording of endogenous calcium and sodium crack channel currents from human T cells. First, FSA garin, a blocker of Sarco endoplasmic reticulum. Calcium ATPase is applied in calcium free extracellular or solution containing three millimolar magnesium to deplete the store.This procedure opens crack channels but does not induce crack current because of the poor permeability of crack channels to magnesium. A whole cell voltage clamp is then established on a T-cell membrane. The cell is perfused with a pipette solution containing a high concentration of the calcium chelator pta.Calcium containing or divalent cation free extracellular solutions are then applied sequentially to activate measurable calcium and sodium currents via crack channels. The resulting data demonstrate the level of functional expression of crack channels in resting human T cells. Investigation of membrane currents via crack channels may provide valuable information on crack channel regulation and functions and establish the role of crack channels in normal immune responses and immunological diseases.Generally, individuals new to this method will struggle due to the small size of human T lymphocytes and low amplitude of endogenous crack currents in these cells. The CD three positive human T lymphocytes for this experiment were obtained from a peripheral blood sample from a healthy volunteer and isolated using tep human T-cell enrichment cocktail and TEP density medium. According to the instructions in the accompanying written protocol, cells were maintained in T-cell culture medium at 37 degrees Celsius in a carbon dioxide incubator.Prepare the following solutions bat solution number one containing three millimolar magnesium and no calcium bat solution Number two, containing 20 millimolar calcium bat solution number three containing 10 millimolar calcium chelator, H-E-D-T-A and no divalent ions and pipette solution containing 12 millimolar calcium chelator bta. Store the solutions at four degrees Celsius for up to one week. On the day of the experiment, pull pipettes with tip diameter of approximately two microns using glass capillaries and a pipette puller.Use silica tubing with filament with the specification shown here and listed in the accompanying text. After pulling the pipette tips are coated with PEC R 61 0 1 semiconductor protective coating and fire polished. Store them in a dust protected place such as a pipette holder.Open the software controlling the patch clamp amplifier here. An E PC 10 patch clamp amplifier is controlled by pulse software. Configure and save the setup on cell and whole cell macros used for giga seal formation and establishing wholesale configuration.Next in the acquisition software, configure and save the stimulation protocol here to record crack currents. The acquisition protocol in the pulse generation window is set as a voltage ramp from minus 120 to plus 100 millivolts of 50 milliseconds. In duration.Create two stimulation protocols with ramp voltage stimulation frequencies of 0.5 hertz and five hertz, and save them in the pulse generation window under the names ramp one and ramp two respectively. To prepare cells for recording plate 500 microliters of the cell suspension containing 0.2 to 0.5 million cells on a recording chamber consisting of a polyol lysine coated cover slip with an attached silicon O-ring. Incubate for 20 minutes at 37 degrees Celsius.Following the incubation, secure the recording chamber in a chamber holder. Here, a custom made chamber holder is used in which the base part supports the cover slip on the bottom of the chamber and the top part presses against the top of the cover slip. Place the chamber holder on the stage of the inverted microscope using a 40 x oil immersion objective and bright field illumination.Find a field of cells and focus. Place the silver silver chloride reference electrode into the bath. Then align a multi barrel gravity driven perfusion system by placing the tip of the inflow tube at 45 degrees to the cover slip close to the field of view, the suction tube is inserted into the top part of the chamber holder.Rotate the top part of the chamber holder until the suction tube is opposite the inflow tube to perform correct channel. Current recordings begin by opening the valve on the perfusion system to perfuse the recording chamber with calcium free. Three millimolar magnesium containing bat solution number one, supplemented with 0.5 to one micromolar AP argon for eight to 10 minutes to block the circa pump.This step depletes the calcium store. Next, use an einor micro loader with a P 20 air displacement pipette. To fill the pipette with the pipette solution, insert the pipette into the holder on the amplifier head stage here.A custom made pressure suction device is connected to pressurized air and the vacuum lines. To create positive or negative pressure in a patch pipette, apply a small positive pressure about three inches of water inside the pipette by connecting the pipette to the pressure suction device outlet via the connective tube. Click the setup control key in the EPC 10 patch clamp window on the computer screen.To execute the setup macro. Find a round cell of an average size with a smooth outer surface that is firmly attached to the cover slip. Then lower the patch electrode into the bath under visual control via the microscope in the oscilloscope window.The current flowing through the pipette can be viewed after immersing the pipette into the bath solution. A step like change in the current amplitude is evoked by the five millivolt voltage test pulse applied to the pipette by the setup macro. The pulse software automatically calculates pipette resistance and displays it in the membrane resistance window.On the computer screen, the pipette resistance is usually five to six mega ohm. Click on the auto control key in the amplifier window on the computer's screen to correct the offset potential generated between the pipette and the reference electrode. The baseline current will shift to a zero current level.Use the micro manipulator to bring the pipette tip in to touch the cell membrane. Once contact is made, there will be a decrease in the amplitude of the current evoked by the test pulse. Apply negative pressure 20 to 40 inches of water to the patch pipette.Monitor the giga seal formation in the oscilloscope window. The amplitude of the current evoked by the test pulse should gradually decrease as the value of the pipette resistance increases to more than five giga ohm. Usually giga seal contact forms quickly, but it may also take one to two minutes to achieve a stable giga seal.Once the giga seal is achieved, disconnect the pipette holder from a pressure suction device by removing the connective tube from the outlet. Click the onsell control key on the computer screen to execute the onsell macro, which was set prior to the experiment. According to the EPC 10 manual, compensate for the pipette capacitance by clicking on the auto control key in the cfat window.In the oscilloscope window, the current transient will disappear and the value of the pipette capacitance will appear in the cfat window to break the cell membrane Inside the pipette, connect the pipette to a 20 cubic centimeter syringe via the connective tube and pull back the plunger to apply additional negative pressure to the pipette. When the cell membrane inside the pipette ruptures two current transients charging the cell membrane to a new voltage level will appear. Click on the whole cell control key on the computer screen to execute the preset whole cell macro.Then to set the holding potential type plus 30 millivolts into the V membrane window, positive holding potential helps prevent calcium dependent crack channel in activation. Next, click on the auto control key in the C slow window. To compensate the cell capacitance in the oscilloscope window, the current transient will disappear.The values for cell capacitance and series resistance will appear in the C slow and R series windows respectively. Series resistance is typically within the range of seven to 20 mega. The C slow values which correspond to values of cell capacitance are typically in the range of 1.5 to 2.5 pico fer.In resting human T cells, click on the ramp one control key in the oscilloscope window to apply a series of voltage ramps with frequency of 0.5 hertz. This will execute the stimulation protocol generated and saved prior to the experiment in the oscilloscope window. The currents evoked by the voltage ramps are shown.Record.These current traces in calcium free solution number one containing three millimolar magnesium. In this solution, the crack current is very small due to the poor permeability of crack channels for magnesium Saban. Use these traces in future analysis as leak currents.The absolute amplitude of the leak at minus 100 millivolts should be equal or less than five pka amps. If the cell is leaky. Stop the experiment and start over with a new patch pipette and a new cell.Next to record calcium current via crack channels switch the perfusion valves to replace bath solution number one with bath solution number two containing 20 millimolar calcium. This allows for calcium ions to flow via crack channels and produce an inward current in the oscilloscope window. An inwardly rectifying calcium current via crack channels develops current amplitude at negative voltages, continues to increase for about one minute due to calcium dependent potentiation of the crack current.Click on the ramp two control key in the oscilloscope window to execute a preset stimulation protocol, which changes the frequency of stimulation with voltage ramps to five hertz increase in frequency of stimulation helps to record maximal sodium current via crack channels, which is transient in nature. Next, switch the perfusion valves again to replace bath solution number two with sodium containing divalent cation free solution number three in the oscilloscope window. A transient larger amplitude inwardly rectifying sodium current via crack channels develops.Once the experiment is over, close the file, it saves the recorded current traces for further analysis to analyze the data, retrieve the saved current records into analysis software. Here pulse software is used for analysis using pairs of cursors in the oscilloscope window of the pulse program. Measure the current amplitudes at the beginning and the end of voltage ramps here.The current amplitudes are measured at minus 100 millivolts and at plus 100 millivolts. Export the values of the current amplitudes into a graphic program such as origin scientific graphing and analysis software. For further analysis and graphical presentation, crack currents were measured in CD three positive resting T cells from a healthy human subject using the method described here, this figure shows a time course of crack currents recorded in whole cell voltage clamp configuration at minus 100 millivolts indicated by the filled circles and plus 100 millivolts indicated by the open circles.Prior to the giga seal formation, the cell was pre incubated for approximately 10 minutes in calcium free. Three millimolar magnesium containing bat solution number one containing 0.5 micromolar orthogon after break-in bat. Solutions were sequentially applied as follows, calcium free three millimolar magnesium containing bat solution number one, followed by 20 millimolar calcium containing bat solution number two, followed by divalent cation free bath solution number three, followed by 20 millimolar calcium containing bath solution Number two, the cell was stimulated with a series of voltage ramps from minus 120 millivolts to plus 100 millivolts as shown here.Voltage ramps were applied every 200 milliseconds or at a frequency of five hertz in divalent cation free bat solution number three and every two seconds or at a frequency of 0.5 hertz in all other solutions. Note the slow development of calcium current via crack channels after application of 20 millimolar calcium containing bat solution number two, and fast transient development of sodium current via crack channels in divalent cation free bat solution number three. This figure shows representative current traces recorded during voltage ramps in calcium free.Three millimolar magnesium containing bat solution number one 20 millimolar calcium containing bat solution number two, and divalent cation free bat solution. Number three, the calcium and sodium current traces during the individual voltage ramps can be obtained by subtracting currents recorded in calcium free three millimolar magnesium containing bat solution number one from currents recorded in 20 millimolar calcium containing bath solution number two, and divalent cation free bath solution Number three, the resulting current traces displaying current voltage relationships typical for calcium and sodium currents via crack. Channels are seen here.Once mastered this technique and all the states of the procedure are performed properly, one can record crack channel currents from five to 10 cells a day. After watching this video, you should have a good understanding of how to record correctional current in rest in human T cells. Following this procedure, you should be also able to record correct currents in other cells of immune systems such as activated human T cells, monocytes, macrophages, and also cell lines.Electrophysiological.Recording of crack channel currents in the cells of immune system may enable researchers to establish their role in calcium homeostasis in immune cells.

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Research

JoVE Journal

Biology

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Functional Characterization of Endogenously Expressed Human RYR1 Variants

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Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

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