Abstract
Transient receptor potential canonical (TRPC)4 and TRPC5 channels are non‐selective calciumpermeable cation channels that maintained by phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and inactivated due to its depletion. Phospholipase C (PLC) is an enzyme that cleaves phospholipids and the PLCδ is the most calcium‐sensitive form among the isozymes that stimulated by physiological concentration of Ca2+. Here, we investigated the regulation mechanism of TRPC4 by the Ca2+‐PLCδ‐PI(4,5)P2 signaling cascade. In order to identify the interaction between ion channel and PLCδ protein, we implied co‐immunoprecipitation and fluorescence imaging including Föster Resonance Energy Transfer. We evaluated the activity of channels with electrophysiological recording in HEK293 cells expressing TRPC channels. Here, we demonstrate that TRPC4 directly interacts with PLCδ1. We also found that PLCδ isozymes are activated by the calcium through opened channels but not by the cytosolic calcium increase. Our study established the PLCδ1 inhibits overall TRPC4’s currents activity, but mainly regulates the basal TRPC4 currents to have a characteristic low basal activity. These regulation of PLCδ1 on TRPC4 activity occurs depend on PI(4,5)P2 hydrolysis. Resultantly, PLCδ1 is considered to be a negative regulator of TRPC4.Support or Funding InformationThis research was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean government (2018R1A4A1023822 to I.S.S.). H.Kang, J.Ko., J.Myeong., and M.Kwak. were supported by the BK plus program from the MSIP.
Highlights
Transient Receptor Potential (TRP) channels are nonselective cation channels which are permeable to Ca2+
By comparing the effect of PLCδ1 on TRPC4β mutant which interacts with PIP2, we suggest that each residue affects the channel activity differently by binding with PIP2
To identify the expression patterns, we performed fluorescence imaging experiments in a Human Embryonic Kidney (HEK)293 cell line transiently co-transfected with ECFP tagged channel (TRPC4β or TRPC5) and EYFP tagged PLCδ (PLCδ1 or PLCδ3) enzyme
Summary
Transient Receptor Potential (TRP) channels are nonselective cation channels which are permeable to Ca2+. Among TRP channels, this canonical family is composed of 7 types, and they are subdivided into two groups based on their amino acid sequence and structures; TRPC1, 4, 5 and TRPC3, 6 ,7. Many researches demonstrated the effect of downstream molecules on TRPC channels, which includes PLC, phosphatidylinositol 4,5-biphosphate (PIP2), diacylglycerol (DAG), protein kinase C (PKC) and Ca2+. We previously emphasized the self-limiting mechanism of Gαq pathway on channels that Gαq protein solely activates TRPC1/4 and TRPC1/5 channels, and PIP2 depletion and PKC or Ca2+ inhibits them. We previously emphasized the self-limiting mechanism of Gαq pathway on channels that Gαq protein solely activates TRPC1/4 and TRPC1/5 channels, and PIP2 depletion and PKC or Ca2+ inhibits them8,9 These data implicate that TRPCs are strongly activated by Gαq, fast and tight regulation is necessary afterwards
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