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

Abstract

Source: Malysz, J. et al. Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents. J. Vis. Exp. (2020)

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 v....

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 r.......

Materials

List of materials used in this article

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