Amphotericin-B Mediated Perforated Patch-Clamp Analysis: An Electrophysiological Technique to Study Ionic Currents in Urinary Bladder Cells

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

1. 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.
2. 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.
3. 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.
4. 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.
5. Pull multiple patch electrodes, fire-polish electrode tips, and (if needed) coat the tips with dental wax.
6. 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.
7. Backfill the electrode with the same pipette solution containing amphotericin-B.
8. Mount the electrode onto a holder connected to a patch-clamp amplifier headstage.
9. Using a micromanipulator, place the electrode just below the surface of the extracellular solution so that the tip of the electrode is just submerged.
10. 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).
11. 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.
12. 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.
13. 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).
14. 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.
15. 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.
16. 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).
17. 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.
    1. 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.
    2. Run the voltage protocol in continuous ~1 min interval during an experiment recording currents for pre-addition control, test condition, and washout

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.

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.

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