Whole-Lysosome Patch Clamp Recordings

Whole-lysosome patch clamp recordings were done as previously described^{51 (link)}. Briefly, transfected cells were re-plated onto 12-mm coverslips ~36 hr after transfection. Lysosomes were enlarged with vacuolin-1 (1 μM, overnight) and were released from cells using glass pipettes. Polished glass pipettes used for patch-clamp recordings had resistance of 5–8 MΩs. Pipette solution (lumen) contained (in mM) 145 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, 10 MES and 10 glucose (pH adjusted to 4.6 with NaOH). Bath solution contained (in mM) 140 K-gluconate, 4 NaCl, 2 MgCl₂, 0.39 CaCl₂, 1 EGTA and 10 HEPES (pH adjusted to 7.2 with KOH). Recordings were done 48 - 72 hr after transfection. Signals were amplified and filtered at 1 KHz using a MultiClamp 700B amplifier, and digitized at 5 KHz with a Digidata 1400A digitizer, both controlled with Clampex 10.4 (from Molecular Devices). Lysosomes were held at 0 mV. Current-voltage relationships were obtained using a ramp protocol from −100 mV to +50 mV in 1 s. Data were analyzed using Clampfit 10.4 (Molecular Devices), Excel (Microsoft) and Origin (Origin Lab).