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