Theta Tube-Based Rapid Solution Exchange

Cells were subjected to a continuous slow bath flow of Ringer’s solution. The fresh external Ringer’s solution was supplied to the chamber by the valve-controlled gravity perfusion system VC3-4CLG from ALA Scientific Instruments (Farmingdale, New York). This supplied solution was simultaneously sucked out by the vacuum pump system DR 4-000-007 from Drummond Scientific Company (Broomall, Pennsylvania). The external Ringer’s solution exchange for drug treatment was accomplished by a theta tube moved laterally by a step-driven motor (SF-77C) from Warner Instruments (Holliston, Massachusetts) and was complete within 20 ms. The external Ringer’s solution used for FRET and confocal imaging contained the following (in mM): 160 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 8 glucose, adjusted to pH 7.4 with NaOH. The pipette solution for FRET under the normal GTP concentration (0.1 mM) condition contained the following (in mM): 175 KCl, 5 MgCl₂, 5 HEPES, 0.1 BAPTA, 3 Na₂ATP, and 0.1 Na₃GTP, adjusted to pH 7.4 with KOH. For the case of the GTP depletion condition, the 0.1 mM Na₃GTP was substituted with 1 mM GDPβS. For the case of the nonhydrolyzable GTP analog experiments, the 0.1 mM GTP was substituted with 0.1 mM GTPγS. When using Oxo-M as an agonist, a concentration of 10 μM was used. Oxo-M, Ach and poly-l-lysine were from Sigma-Aldrich (St. Louis, Missouri). DMEM, Lipofectamine-2000, and penicillin/streptomycin were from Invitrogen (Waltham, Massachusetts). Fetal bovine serum was from Gemini Bio-Products (West Sacramento, California). YM-254890 was from Cayman Chemical (Ann Arbor, Michigan).