Shm 828

Temperature control for both calcium imaging and patch clamping was achieved by perfusion of preheated solutions as described previously13 (link). Briefly, solutions were heated with an SHM-828 eight-line heater controlled by a CL-100 temperature controller (Harvard Apparatus). The patch pipette was placed about 1 mm from the solution output ports. Local temperature was accurately monitored by a BAT-12 thermometer with ultrafine thermocouple probe (Physitemp) placed right next to the pipette. The thermometer’s temperature readout was fed into an analog input of the patch amplifier and recorded simultaneously with current in patch clamp recordings. For calcium imaging, MetaMorph software was synchronized with PatchMaster software to record temperature simultaneously with imaging. When the experimental temperature was not controlled, recordings were conducted at room temperature at ~24 °C.

To heat the membrane patch containing ion channels, the bath solution heated by an SHM-828 eight-line heater driven by a CL-100 temperature controller (Harvard Apparatus, Holliston, MA) was perfused to the pipette tip. A custom-made manifold was attached to the output ports of the heater to increase flow volume and to provide heat insulation. To accurately monitor local temperature at the pipette tip, we placed the IT-24P ultrafine thermocouple bead of a BAT-12 microprobe thermometer (Physitemp, Clifton, NJ) less than 1 mm from the pipette tip. HEKA patch-clamp amplifier registered temperature readings from the thermometer simultaneously with current recording. The speed of temperature change was set at a moderate rate of about 0.3°C/s. This rate ensured that heat-driven gating transitions of the channels reached equilibrium during the course of temperature change, so that the channels were recorded at the equilibrium state. When the experimental temperature was not controlled, recordings were conducted at room temperature at 24°C.