Pclamp software suite

Following break-in, mEPSCs were recorded for 15–20 min at a holding potential of −70 mV in the presence of 1 μM tetrodotoxin, which blocks evoked synaptic transmission. Only mEPSCs beginning 5 min after break-in were analysed. Recordings were rejected if the holding current or series resistance varied by more than 25% during the recording. Samples are represented by the total number of cells from more than five mice per group. Raw data were analysed using Clampfit (pClamp software suite version 10.2; Molecular Devices). Statistical analysis was performed using GraphPad Prism.

CosM6 cells were transfected with 1–2 μg of wild type or truncated constructs using FuGENE6 (Promega) and used for patching within 1–2 days after transfection. Symmetric internal potassium (K_int, 140 mM KCl, 1 mM EGTA, 1 mM K₂EDTA, 4 mM K₂HPO₄, pH 7.38) or cesium (Cs_int, 150 mM CsCl, 1 mM EGTA, 1 mM K₂EDTA, 5 mM Hepes, pH 7.38) buffers were used for single-channel recordings. Upon patch excision in the inside-out configuration, the bath was perfused with K_int or Cs_int containing GSK101 (10 nM to 5μM), so that the drug was applied from the cytoplasmic side of the membrane. For experiments with divalent ions (Ca²⁺ and Ba²⁺), cells were patched in symmetric buffer (75 mM CaCl₂ or BaCl₂ respectively with 5 mM Hepes, pH 7.3). Upon patch excision the bath was perfused with K_int including 10 nM of GSK101. Recordings were made and digitized with the Axopatch 1D patch-clamp amplifier and the Digidata 1200 digitizer (Molecular Devices). Data were collected at 10 kHz, low-pass filtered at 5 kHz and analyzed with the pClamp software suite (Molecular Devices). The pipettes with bubble number (BN)^{84 (link)} 4.5 – 5.0 (~ 2 M Ohm in symmetric K_int) were fabricated from the Kimble Chase soda lime glass with a Sutter P-86 puller (Sutter Instruments). All measurements were carried out at −30 mV membrane potential or as specified in the text.