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 (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.
1. Preparation of Resting Human T Lymphocytes
2. Preparation of the Recording Chamber
3. Preparation of Patch Pipettes
4. Patch Clamp Setup Preparation
Chemicals | Bath Solutions (mM) | Pipette Solutions (mM) | ||
# 1 | # 2 | # 3 | ||
CH3SO3Na | 130 | 110 | 125 | – |
NaCl | 2 | 4 | 5 | – |
Ca(OH)2 | – | 20 | – | – |
MgCl2 | 3 | 1 | – | 5 |
MgSO4 | – | – | – | 2 |
HEPES | 10 | 10 | 10 | 15 |
HEDTA | – | 10 | – | |
EDTA | – | – | 1 | – |
Cs-aspartate | – | – | – | 125 |
BAPTA | – | – | – | 12 |
Glucose | 10 | 10 | 10 | – |
Table 1. Solutions for whole-cell CARC current recordings.
All chemicals were purchased from Sigma-Aldrich, St. Louis, MO.
5. Mounting Cells on the Microscope Stage
6. CRAC Channel Currents Recording
7. Data Analysis
8. Representative Results
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).
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 μm). 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 resting T cells to better understand the nature of normal and pathological immune cell responses. Using this protocol, one can measure CRAC currents in resting human T cells as well as in other immune cells, such as activated human T cells, monocytes, and macrophages.
The authors have nothing to disclose.
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.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
RosetteSep Human T Cell Enrichment Cocktail | StemCell Technologies, Vancouver, BC, Canada | 15061 | ||
RosetteSep Density Medium | StemCell Technologies | 15705 | ||
RPMI-in 1640 medium w/glutamine/HEPES | Fisher, Waltham, MA | SH3025501 | ||
Fetal Calf Serum | Omega Scientific, Tarzana, CA | FB-01 | ||
GlutaMAX-I (100X solution) | Invitrogen, Carlsbad, CA | 35050 | ||
RPMI 1640 vitamin solution (100X) | Sigma-Aldrich | 7256 | ||
1640 amino acids solution (50X) | Sigma-Aldrich | R7131 | ||
Sodium pyruvate | Sigma-Aldrich | S8636 | ||
β-Mercaptoethanol | Sigma-Aldrich | M7522 | ||
Inositol trisphosphate | Sigma-Aldrich | 19766 | ||
BAPTA | Sigma-Aldrich | A4926 | ||
Poly-L-Lysine Hydrobromide | Sigma-Aldrich | P2636 | ||
Lanthanum Chloride | Sigma-Aldrich | 262072 | ||
Thapsigargin | Calbiochem | 586005 | ||
Sylgard 184 Silicon Elastomer Kit | Dow Corning, Midland, MI | 3097358-1004 | ||
HIPEC R6101 Semiconductor Protective Coating | Dow Corning, Midland, MI | |||
63-500 Series High-Performance Vibration Isolation Lab Table | Technical Manufacturing, Peabody, MA | 63-540 | ||
EPC 10 patch clamp amplifier with headstage | HEKA Instruments, Bellmore, NY | |||
Micromanipulator | Sutter Instrument, Novato, CA | MP-285 | ||
Olympus 1X71 Inverted microscope with 40x oil immersion objective | Olympus America, Center Valley, PA | 1X71 | ||
Windows Computer | Dell | |||
Pulse software | HEKA Instruments | |||
Origin Scientific Graphing and Analysis Software | OriginLab, Northampton, MA | |||
Patch pipette puller | Sutter Instrument | P-97 | ||
Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm | Sutter Instrument | BF150-110-7.5 | ||
Narashige’s Microforge | Tritech Research, Los Angeles, CA | MF-830 | ||
Silicon O-rings | McMASTER-CARR, Santa Fe Springs, CA | 111 S70 | ||
Coverslips 25 mm | Fisher Scientific | 12-545-102 25 mm 25CIR.-1 |