Pclamp v 10

The cells were transferred onto a small chamber on the stage of an inverted microscope (IX70, OLYMPUS, Tokyo, Japan), and attached to coverslip in the small chamber for 10 min prior for the recording. Transiently transfected cells were identified by their fluorescence tagging. Recording pipettes were pulled from glass capillaries (Harvard Apparatus, Holliston, MA, USA) using puller (PC-10, NARISHIGE, Tokyo, Japan). Whole-cell currents were recorded using Axopatch 200B amplifier (Molecular Devices, Foster City, CA, USA) and Digidata 1550B interface (Molecular Devices). Experiments were performed at room temperature (18-22˚C). The recording chamber was continuously perfused at a flow rate of 1-2 ml/min. Glass microelectrodes with 2-2.5 megaohms resistance were used to obtain gigaohm seals. The whole cell configuration was used to measure the TRPC4β channel currents in HEK293 cells. Voltage ramps ranging from +100 to -100 mV over period of 500 ms were imposed every 10 s with a holding membrane potential of -60 mV. pCLAMP v.10.2 (OriginLab, Northampton, MA, USA) was used for data acquisition and the data were analyzed using the OriginPro 8 (OriginLab).