Overview
This video demonstrates the characterization of cationic currents from transient receptor potential melastatin type 4 (TRPM4) ion channels in detrusor smooth muscle cells using amphotericin-B mediated perforated patch-clamp technique. The technique helps evaluate the biophysical and pharmacological properties of ion channels.
Protocol
1. Recording Voltage-step Induced Cation Currents from DSM Cells Using Amphotericin-B Perforated Whole-cell Voltage Patch-Clamp Technique
- Pipette 0.25–1 mL of cell suspension onto a glass-bottom chamber sitting on a stage of an inverted microscope and allow the cells to adhere to the glass bottom.
- After incubation for at least 45 min, remove digestion solution (DS), from the bath and replace with the E solution (Table 1) by superfusion where the gravity-aided-solution flow via inlet tubing slowly replaces DS with the new solution while outlet tubing connected to a vacuum waste vessel removes the chamber solution and prevents overflow.
NOTE: E solution contains tetraethylammonium (TEA+) and cesium (Cs+) ions to inhibit K+ currents. - Prepare a working stock solution of amphotericin-B in dimethyl sulfoxide (DMSO) (1 mg per 10 µL of DMSO). To fully dissolve amphotericin powder, sonicate (at least 15 min) and vortex the solution well.
NOTE: This step usually takes less than 10 min. Dissolving 3–4 mg of amphotericin-B in 30–40 µL of DSMO in a 1.5 mL microcentrifuge tube works well. Higher quantities of amphotericin-B require more DSMO solvent typically resulting in a longer interval for mixing and incomplete solubilization of the amphotericin-B solid particles present in the tube. - Dissolve stock solution of amphotericin-B in the pipette solution (solution P, Table 1) to obtain a final concentration of 200–500 µg/mL. This step requires extensive sonication and vortexing at a high-speed setting (8–10/10) for ~30 to 60 min per step to ensure optimal mixing and prevention of amphotericin-B precipitate formation in the pipette solution.
NOTE: Amphotericin-B will precipitate over time and is light-sensitive. The working pipette solution containing Amphotericin-B is checked for solubility, hand-mixed prior to pipette filling, and kept in the dark. - Pull multiple patch electrodes, fire-polish electrode tips, and (if needed) coat the tips with dental wax.
- Fill the tip of a patch electrode with the pipette solution (solution P, Table 1) without amphotericin-B by briefly dipping the electrode in the solution.
- Backfill the electrode with the same pipette solution containing amphotericin-B.
- Mount the electrode onto a holder connected to a patch-clamp amplifier headstage.
- Using a micromanipulator, place the electrode just below the surface of the extracellular solution so that the tip of the electrode is just submerged.
- In the voltage-clamp mode, set the holding potential to 0 mV and adjust the current to 0 pA with the pipette offset dial on the commercial amplifier (Table of Materials).
- Determine the electrode resistance using the Membrane Test window/function of the commercial acquisition software (Table of Materials). To activate click Tools>Membrane Test>Play or a shortcut icon in the software. The determined electrode resistance should be in the range of 2 to 5 MΩ.
NOTE: Membrane Test function provided in the commercial acquisition software or Seal Test option on the amplifier can be used to monitor electrode resistance by applying voltage steps repetitively. - Continue monitoring electrode resistance while advancing the electrode toward a chosen DSM cell with a micromanipulator (Figure 1A).
NOTE: To be considered a viable DSM cell, the cell must show spindle-shaped elongated morphology, a well-defined halo around the cell, crisp edges, and semi-contractile (serpentine) appearance. - When touching the cell surface with the electrode—indicated by a rapid increase in the electrode resistance measured with the Membrane Test function—form a giga-seal by applying gentle rapid negative pressure to the electrode holder via tubing. This results in negative pressure created at the tip of the electrode that pulls cell membrane into electrode aiding in the formation of a giga-seal or a very tight contact between the electrode and plasma membrane (Figure 1B).
- Once the giga-seal forms, compensate pipette capacitance by adjusting fast and slow dials on the commercial amplifier and monitor giga-seal stability (leak current) using the Membrane Test function.
- Allow time, typically 30–60 min, for amphotericin-B to diffuse down the pipette and be inserted into the plasma membrane forming pores primarily selective to monovalent cations. During this step, continue monitoring the giga-seal with the Membrane Test function. As cell perforation increases so does the amplitude of the capacitance transients (compare Figure 1B versus Figure 1C displaying no and effective cell perforation, respectively) measured with the Membrane Test function.
- When the patch perforation is optimal (judged by stable series resistance typically below 50 MΩ), cancel out the capacitance transients by adjusting the dials for cell capacitance and series resistance on the amplifier. Series resistance compensation can also be performed at this time (Figure 1D).
- Once stable voltage-step induced cation currents evoked by the specified protocol are observed, apply a compound or physiological condition to test by superfusion and record the responses for the control-, test-condition, and washout (if possible) with the commercial acquisition software.
- Record currents with a routine voltage-step protocol that involves holding DSM cells at -64 or -74 mV and stepping the voltage in 10 mV increments for 400 or 500 ms from -94 to +96 or +106 mV and returning to the holding potential.
NOTE: The membrane potential values are adjusted for a liquid junction potential of 14 mV (using P and E solutions, Table 1). The liquid junction potential is obtained in the commercial acquisition software (Table of Materials) by clicking Tools>Junction Potentials and entering the concentrations of solution ion components. A ramp protocol can also be used to obtain current recordings. - Run the voltage protocol in continuous ~1 min interval during an experiment recording currents for pre-addition control, test condition, and washout
- Record currents with a routine voltage-step protocol that involves holding DSM cells at -64 or -74 mV and stepping the voltage in 10 mV increments for 400 or 500 ms from -94 to +96 or +106 mV and returning to the holding potential.
Solution Type | Composition (in mM) |
DS (Dissection/ Digestion Solution) | 80 Na-glutamate, 55 NaCl, 6 KCl, 10 HEPES, 2 MgCl2, and 11 glucose, pH adjusted to 7.4 (with 10 M NaOH) |
DS-P (Papain-containing DS) | DS containing 1–2 mg/ml papain, 1 mg/ml dithiothreitol and 1 mg/ml bovine serum albumin |
DS-C (Collagenase-containing DS) | DS solution containing 1–2 mg/ml collagenase type II, 1 mg/ml bovine serum albumin, 0 or 1 mg/ml trypsin inhibitor and 100-200 μM Ca2+ |
P (Pipette) | 110 CsOH, 110 aspartic acid, 10 NaCl, 1 MgCl2, 10 HEPES, 0.05 EGTA, and 30 CsCl,pH adjusted to 7.2 with CsOH, and supplemented with amphotericin-B (300-500 μg/ml) |
E (Extracellular) | 10 tetraethylammonium chloride (TEA), 6 CsCl, 124 NaCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.3–7.4 with NaOH or CsOH, and 0.002–3 (2–3 mM) nifedipine |
Table 1: Compositions of dissection/digestion solution (DS), and pipette and extracellular solutions used in perforated patch-clamp experiments.
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Representative Results
Figure 1: Schematic illustration of steps involved in giga-seal formation and amphotericin-B perforation of human DSM cells. Illustrated are spatial positions of an amphotericin-B containing pipette and a DSM cell along with associated responses for membrane tests obtained in the commercial acquisition software (Table of Materials) by altering voltage steps (either -10 or -20 mV in this example) determining resistance. Configurations are: (A) prior to the cell approach with an electrode, (B) after giga-seal formation obtained by positioning the amphotericin-B containing pipette (amphotericin-B represented by red dots) onto the cell surface and applying negative pressure, (C) the on-cell configuration shown ~45 min after giga-seal formation, at this time-point amphotericin-B has diffused down the pipette and its molecules have inserted into the plasma membrane at the tip of the electrode forming cation permeable pores, and (D) the same configuration as in (C) but with capacitance transients canceled out using dials for whole-cell capacitance and series resistance on the amplifier.
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Materials
Name | Company | Catalog Number | Comments |
5 ml polystyrene round-bottom tube | Falcon | 352054 | Tubes for DS containing enzymes used in digestion steps |
Amphotericin-B | Fisher | BP928-250 | Used for patch/cell perforation |
Amphotericin-B | European Pharmacopoeia Reference Standards | 5 | Used for patch/cell perforation |
Amphotericin-B | Sigma-Aldrich | A9528-100MG | Used for patch/cell perforation |
Analog vortex mixer | VWR | 58816-121 | |
Aspartic acid | Sigma-Aldrich | A9006 | Intracellular pipette solution |
Bovine serum albumin | Sigma-Aldrich | A7906 | DS |
CaCl2 | Sigma-Aldrich | C1016 | Extracellular solution and DS |
Capillary Glass | Sutter | BF150-110-7.5 | Capillary for preparation of pulled patch electrodes |
Cesium hydroxide hydrate | Sigma-Aldrich | C8518 | Intracellular pipette solution |
Clampex ver. 10 software includes data acqusition (Clampex) and analysis (Clampfit) programs | Axon Instruments/ Molecular Devices | pCLAMP-10 | Commerical software and part of patch-clamp rig setup |
CsCl | Sigma-Aldrich | 203025 | Extracellular and intracellular solutions |
Dental wax | Miltex Dental Wax Technologies, Inc. | 18058351 | |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650 | Solvent |
EGTA | Sigma-Aldrich | E3889 | Ca2+ chelator, used in intracellular pipette solution |
Flaming/Brown micropipette puller | Sutter | P-97 | Required to pull electrodes with very fine tips |
Floating foam tube rack/holder | VWR Scientific | 82017-634 | Used for holding tubes with enzymes for temperature control |
Glucose | Sigma | G8270 | |
Glutamic acid (Na salt) | Sigma-Aldrich | G1626 | DS |
HEPES | Sigma-Aldrich | H3375 | pH Buffer |
KCl | Fisher Scientific | BP366-1 | Extracellular solution |
Low Noise Data Acquisition System | Axon Instruments/ Molecular Devices | Digidata 1440A | Part of patch-clamp rig setup |
Magnetic stirrer | VWR | 01-442-684 | |
MgCl2 (hexahydrate) | Sigma-Aldrich | M2670 | Extracellular and intracellular solutions |
MicroForge | Narishige | MF-830 | Used for fire-polishing electrodes |
NaCl | Sigma-Aldrich | S7653 | Extracellular and intracellular solutions |
NaOH | Sigma-Aldrich | S8045 | |
Nifedipine | Sigma-Aldrich | N7634 | L-type voltage-gated Ca2+ channel blocker |
Nikon inverted microscope, TS100 with T1-SM stage with 5x, 10x, 20x, and 40x objectives | Nikon | Discontinued | Part of Patch-clamp rig setup |
Non-metalic syringe needle, MicroFil | WPI | MF-34G-5 | Filling of intracellular pipette solution |
Pasteur pipette | FisherBrand | 13-678-20A | Tips are broken off and fire-polished and used for titration of enzymatically treated tissues to release single DSM cells from pieces |
Patch-clamp amplifier | Axon Instruments/ Molecular Devices | Axon Axopatch 200B | Part of patch-clamp rig setup |
PC computer | DELL | Custom configuration | Part of patch-clamp rig setup |
pH Meter | Aspera Instruments | PH700 | |
Polyethylene tubing | Intramedic | 427-436 | Tubing for superfusion of extracellular bath connected to glass-bottom recording chamber |
Tetraethylammonium chloride | Sigma-Aldrich | T2265 | Ion channel blocker of Kv and BK channels added to the extracellular bath solution |
Weighting scale | Mettler Toledo | XS64 | |
ZeissAxiovert 40C inverted microscope with 10x and 40x objectives | Carl-Zeiss | Discontinued | Part of patch-clamp rig setup |