Perforated patch recordings of neuronal calcium currents (ICa) with amphotericin B or nystatin reduce dialysis of intracellular constituents and current rundown, but can be difficult and frequently unsuccessful. We investigated the saponin β-escin as a putative ionophore for perforated patch ICa recordings in acutely dissociated, rat dorsal root ganglion neurons. ICa was recorded in time-course studies after including either β-escin (50 μM), or amphotericin B (240 μg/ml) as perforating ionophores in the internal pipette solution, in comparison to standard ruptured-patch technique, using suction. Perforated patches were allowed to take place spontaneously. The percentage loss of ICa per min (within the first 20 min) was significantly less after β-escin (0.0518%) (n=18), versus either amphotericin (1.82%) (n=12) or standard patch (4.52%) (n=7), (P<0.001). The slope of the rundown after linear fit was also less after β-escin (P<0.001). Minimal "steady-state" access resistance (Ra) of 6.5±1.6 MΩ was achieved within 7.1±9.3 min following perforation with β-escin, 7.9±3.5 MΩ within 44±14 min after amphotericin B, and 6.8±1.9 MΩ with standard patch (P<0.05 for Ra, and P<0.01 for permeabilization time, respectively). Success rates were 59% with β-escin versus 27% with amphotericin. Leak >10% of peak ICa was present in 25% of cells after β-escin versus 20% after amphotericin, and 12% after standard technique. Perforated patches using β-escin were stable for 15-60 min. We conclude that β-escin may be used as an alternative ionophore for perforated patch-clamp studies in neurons, and results in minimal rundown that can facilitate long-term recordings of ICa. Limited rundown may be due to better preservation of cytosolic ATP content.
- Amphotericin B
- Calcium current: voltage-gated
- Current rundown
- Dorsal root ganglion
- Patch-clamp: whole-cell, perforated
ASJC Scopus subject areas