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

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

Table 1: Compositions of dissection/digestion solution (DS), and pipette and extracellular solutions used in perforated patch-clamp experiments.