The classic transient receptor potential channel (TRPC) is likely to be a Ca 2ϩ -permeable cation channel in mammalian cells. Five different TRPC channels have been isolated. Among them, TRPC5 was initially hypothesized to be a store-operated channel (SOC) 1) ; however, it was then shown to be activated by stimulating G protein-coupled receptors (GPCRs). Therefore, TRPC5 was suggested to be a receptoroperated channel (ROC).2-5) TRPC4 and TRPC5 are rapidly desensitized after activation by GPCR. This desensitization does not depend on extracellular monovalent cations, such as Na ϩ or Cs ϩ . TRPC4 and TRPC5 are desensitized after the activation of muscarinic receptors. Even when intracellular GTPgS has been used to activate TRPC4 and TRPC5, their currents are rapidly desensitized.Activated G proteins stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) through PLCb. PIP 2 is then hydrolyzed into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). Among mammalian TRPs, TRP vanilloid 1 (TRPV1) has been shown to be inactivated by PIP 2 . 6,7) Alternatively, PIP 2 activates TRP melastatin 5 (TRPM5), 8) TRPM7, 9) TRPM8, 10) TRPV5, 11) and TRPC6. 12) In the TRPM4 channel, current was recovered by PIP 2 after an initial decay.
13)Previously, we showed that TRPC5 is likely to be a nonselective cationic currents (NSCC) channel, activated by muscarinic receptor stimulation. 3,14,15) In subsequent studies, we showed that TRPC5 has two regulatory NSCC pathways activated by stimulation of muscarinic receptors in native tissues 16,17) : the DAG-protein kinase C (PKC) negative feedback pathway, [16][17][18] and the calmodulin (CaM)-myosin light chain kinase (MLCK) positive feedback pathway. [19][20][21] To further confirm the identity of TRPC5 as an NSCC activated by muscarinic receptor stimulation, we investigated the effects of intracellular PIP 2 on TRPC5 channels expressed in human embryonic kidney (HEK) cells. We found that the desensitization of TRPC5 current is associated with both PIP 2 and PKC. 17) We also determined that the actin cytoskeleton is involved in the activation and desensitization of TRPC5 currents. Additionally, in mouse ileal myocytes, PIP 2 is associated with the desensitization of carbachol (CCh)-activated inward currents. These results give further evidence that TRPC5 is a candidate NSCC channel, activated by muscarinic receptor stimulation.
MATERIALS AND METHODS
Cell Culture and Transient TransfectionHuman embryonic kidney (HEK)293 cells (ATCC, Manassas, VA, U.S.A.) were maintained according to the recommendations of the supplier. For transient transfection, cells were seeded in 6-well plates. The following day, 1 mg/well pcDNA plasmid vectors containing the cDNA for TRPC5-GFP (given kindly by Dr. Shuji Kaneko) and muscarinic receptor 3 were transfected into cells using the transfection reagent FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's protocol. After 24-48 h, cells were trypsinized and used for whole-cell recording.Dissociation of Single C...