Electrophysiological analysis and Ca2+ imaging was carried out as previously described . Briefly, cells were plated on coverslips, placed in a chamber, and perfused on the stage of an upright, fixed-stage microscope (for electrophysiology, model BHWI; Olympus, Lake Success, NY, http://www.olympus-america.com; for imaging experiments, model E600FN; Nikon, Melville, NY, http://www.nikonusa.com) with an oxygenated solution containing NaCl, 140 mM; KCl, 5 mM; CaCl2, 2 mM; MgCl2, 1 mM; HEPES, 10 mM; glucose, 10 mM (pH 7.4). Experiments were performed at room temperature. For whole-cell recording, patch pipettes were pulled on a vertical puller (model PB-7; Narishige, Tokyo, Japan) from borosilicate glass pipettes 1.2 mm outer diameter, 0.95 mm inner diameter; with internal filament (World Precision Instruments, Sarasota, FL, http://www.wisent.ca) and had tips of 1–2 μm outer diameter with tip resistances of 6–12 MΩ. Pipettes were filled with a bathing solution containing KCH3SO4, 98 mM; KCl, 44 mM; NaCl, 3 mM; HEPES, 5 mM; EGTA, 3 mM; MgCl2, 3 mM; CaCl2, 1 mM; glucose, 2 mM; Mg-adenosine triphosphate (ATP), 1 mM; guanosine triphosphate (GTP), 1 mM; and reduced glutathione, 1 mM (pH 7.2). For Ca2+ imaging studies, cells were incubated for 45 minutes in Fura-2/AM (10 μM; Molecular Probes, Eugene, OR) plus nonionic detergent (10 μM; Pluronic F127; Biotium, Hayward, CA). After loading, cells were rinsed (1 mL/minutes) for 30 minutes by perfusion with the bathing solution. Test solutions were applied by bath perfusion. Cells were viewed through a 60× water-immersion objective (1.0 numerical aperture [NA]; Nikon), and fluorescence was stimulated with a 150-W xenon lamp attached to the microscope's epifluorescence port by a liquid light guide (Sutter Instruments, San Raphael, CA), and excitation wavelengths (340 or 380 nm) were changed with a filter wheel (Lambda 10–2; Sutter Instruments). Fluorescence changes were monitored with a cooled charge-coupled device (CCD) camera (SensiCam; Cooke Corp., Auburn Hills, MI, using Imaging Workbench software; Axon, Inc., Burlingame, CA). Images were acquired every 5–10 seconds, using 2 × 2 binning with 0.5–1-second acquisition times.