Practical procedures

Similar to stretch sensitivity, volume/osmosensitivity ofTRP channels may be assessed either in cells endogenously expressing the channel in question or after channel expression in a cell type with low endogenous TRP levels such as HEK 293 cells. For measurements of the volume sensitivity of TRP channels in the whole cell patch clamp mode in mammalian cells, we generally employ an isotonic solution containing (in mM) 105 NaCl, 6 CsCl, 5 CaCl2, 1 MgCl2, 10 HEPES, 90 D-mannitol, 10 glucose, buffered pH 7.4 with NaOH (320 ± 5 mOsm). Cell swelling can thus be induced at constant extracellular ionic strength by omitting the desired amount of mannitol from this solution. The pipette solution for such experiments is generally composed of (in mM) 20 CsCl, 100 Asp, 1 MgCl2, 10 HEPES, 4 Na2ATP, 10 BAPTA, and an equivalent amount of CaCl2 to buffer [Ca2+]; at an required level (for calculation of free intracellular divalent concentrations, we refer to the CaBuf program ftp://ftp.cc.kuleuven.ac.be/ pub/droogmans/cabuf.zip).

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