Hematocrit capillary glass

Patch‐clamp pipettes were prepared from hematocrit capillary glass (VWR Scientific, Radnor, PA) using a vertical puller (David Kopf Instrument, 700C) modified to pull in three stages. They had resistances of 5–8 MΩ. The pipette solution contained (in mmol/L): 140 KCl, 11 EGTA, 2 MgCl₂, 1CaCl_2, 10 NaCl, 2 MgATP, and 10 HEPES, with pH adjusted to 7.4 with N‐methyl‐D‐glucamine (NMDG). The bath solution contained (in mmol/L): 135 NaCl, 2 CaCl₂, 1 MgCl₂, 5 KCl, 10 glucose, and 10 HEPES with pH adjusted to various values with NMDG. Modifications to these basic solutions are described in the text. Changes in extracellular K were made by substitution with Na. Reductions in pipette Cl⁻ were made by substitution with aspartate. Solutions were rapidly changed during recordings using gravity‐fed flow pipes positioned near the cell. Whole‐cell currents were recorded with an EPC‐7 patch‐clamp amplifier (HEKA) and digitized with a Digidata 1332A interface (Molecular Devices, Sunnyvale, CA). We did not use series‐resistance compensation as in the absence of activation of voltage‐gated channels the input resistance of the cell (~500 MΩ) was much larger than the pipette resistance. All recordings were performed at room temperature. Data were analyzed using Clampfit software (Molecular Devices).