An Essential Role of PI(4,5)P2 for Maintaining the Activity of the Transient Receptor Potential Canonical (TRPC)4 Channel TRPC4β

Abstract

The Transient Receptor Potential Canonical 4 (TRPC4) channel is a Ca2+-permeable, non-selective cation channel in mammalian cells and mediates a number of cellular functions. Many studies show that TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. However, our previous study showed that the TRPC4 current was inhibited by a constitutively active form of Gαq(GαqQ209L). It may have caused a shortage of phosphoinositide phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) because GαqQ209L would have persistently activated PLCβ. Therefore, we used an inducible system to regulate PI(4,5)P2 specifically and acutely. The TRPC4β current was reduced by inducible GαqQ209L but not by the mutants whose binding ability to PLCβ is impaired. If the aforementioned phenomenon resulted from desensitization of TRPC4 by protein kinase C (PKC), the current of TRPC4β (T877A) mutant which is not phosphorylated by PKC would not be inhibited by co-expression of a GαqQ209L. However, we detected the inhibitory action of GαqQ209L. Depletion of PI(4,5)P2 using the inositol polyphosphate 5-phosphatase (Inp54p) inducible system led to an irreversible inhibition of TRPC4 currents after application of rapamycin to HEK293 cells co-expressing TRPC4 with Inp54p. On the other hand, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) inducible system did not activate the initial gating of TRPC4β. Even in the case of Gαi2-activated TRPC4β currents, the acute depletion of PI(4,5)P2 led to reduced TRPC4β currents. Therefore, we suggested that PI(4,5)P2 is not the activator for TRPC4 activation but it is still necessary for regulating TRPC4 activation. Especially, TRPC4 desensitization might be a result of hydrolysis of PI(4,5)P2 since TRPC4 desensitization through muscarinic receptor 3 which activates Gαq-PLC pathway disappeared by adding PI(4,5)P2 and nonhydrolysis PI(4,5)P2. These findings indicate an essential role of PI(4,5)P2 for maintaining the activity of TRPC4β.