HEK-293T cells were transfected with 0.2 μg of channel cDNA and 0.04 μg of eYFP cDNA to identify transfected cells. For some experiments, 0.8 μg of m2R and 0.8 μg of m-Phos cDNA were also transfected. Whole-cell patch clamp recordings were performed 24−72 hr after transfection. Borosilicate glass electrodes (Warner Instruments) of 5−7 mΩ were filled with intracellular solution (130 mM KCl, 20 mM NaCl, 5 mM EGTA, 2.56 mM K2ATP, 5.46 mM MgCl2, 0.30 mM Li2GTP, and 10 mM HEPES, pH 7.4). Extracellular 20K solution contained 20mM KCl, 140mM NaCl, 0.5mM CaCl2, 2mM MgCl2 and 10mM HEPES (pH7.4). Alcohols (0.1 − 300 mM), carbachol (5 μM), or BaCl2 (1 mM) were diluted into the 20K solution and applied directly to the cell with the rapid valve-controlled, perfusion system (Warner Instruments, VC6, MM-6 manifold). All chemicals for electrophysiology were purchased from Sigma-Aldrich. Whole-cell patch-clamp currents were recorded using Axopatch 200B (Molecular Devices; Axon Instruments) amplifier. Currents were adjusted electronically for cell capacitance and series resistance (80−100%), filtered at 1kHz with an 8-pole Bessel filter and digitized at 5kHz with a
