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

Perforated patch-clamp technique allows real-time measurements of ionic currents associated with the activity of ion channels on cells like detrusor smooth muscle, or DSM, cells of the urinary bladder.

To begin, load the DSM cell suspension onto a glass chamber and incubate to allow cell adherence. Next, add an extracellular solution containing tetraethylammonium and cesium ions to the adhered cells. These cations block potassium channels on the cell membrane and inhibit the interfering potassium currents.

Subsequently, take a polished patch-clamp pipette - a thin hollow glass tube containing a recording electrode connected to an amplifier. Front fill the pipette with an intracellular solution and backfill it with the same solution but containing amphotericin-B, a membrane perforating antibiotic.

Now, submerge the pipette tip into the extracellular solution containing DSM cells. After setting the potential and current to appropriate levels, advance the pipette tip towards the cell membrane and simultaneously monitor the electrode resistance.

A rapid increase in the resistance value indicates that the pipette tip has reached the cell membrane. Now, apply gentle suction to the electrode to form a tight seal called giga-seal between the pipette tip and the plasma membrane.

Following seal formation, amphotericin slowly diffuses into the tip and perforates the membrane, forming ion pores. Once optimum perforation is achieved, ions move between the cells and the electrode, generating currents proportional to ion channel function.

Pipette 0.25 to 1 milliliter of cell suspension onto a glass-bottom chamber sitting on the stage of an inverted microscope and incubate it for at least 45 minutes to allow the cells to adhere. Then, remove the DS from the bath and replace it with E solution via superfusion.

Pull multiple patch electrodes, fire-polish the electrode tips, and coat the tips in dental wax if needed. Fill the tip of a patch electrode with the pipette solution without amphotericin-B by briefly dipping the electrode in the solution. Success of patch-clamp electrophysiology depends on DSM cell quality and amphotericin-B solubilization.

Backfill the electrode with the same pipette solution containing amphotericin-B and mount the electrode onto a holder connected to a patch-clamp amplifier head stage. Use a micromanipulator to place the electrode just below the surface of the extracellular solution so that the tip of the electrode is just submerged.

Then, determine the electrode resistance using the Membrane Test window function of the commercial acquisition software and advance the electrode toward the cell of interest. When touching the cell surface with the electrode, form a giga-seal by applying gentle rapid negative pressure to the electrode via tubing. This results in negative pressure at the tip of the electrode, resulting in a successful giga-seal as confirmed by the Membrane Test.

Allow 30 to 60 minutes for the amphotericin-B to diffuse down the pipette and be inserted into the plasma membrane, forming pores primarily selective to monovalent cations. Continue monitoring the giga-seal with the Membrane Test function.

When the patch perforation is optimal, cancel out the capacitance transients by adjusting the dials for cell capacitance and series resistance on the amplifier. Once the stable voltage-step cation currents are observed, record currents with the routing voltage-step protocol as described in the manuscript.