Korean J Physiol Pharmacol.  2023 Mar;27(2):187-196. 10.4196/kjpp.2023.27.2.187.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1

• Ko J
• Kim J
• Myeong J
• Kwak M
• So I

• KMID: 2539573
• DOI: http://doi.org/10.4196/kjpp.2023.27.2.187

Abstract

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4β is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP₂ ). In this study, we present the regulation mechanism of the TRPC4 channel with PIP₂ hydrolysis which is mediated by a channel-bound PLCδ1 but not by the G_qPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca²⁺ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP₂ depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP₂ level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca²⁺ ions into the cell and promoting the PIP₂ hydrolyzing activity of PLCδ1.

Keyword

Calcium; Fluorescence resonance energy transfer; Phosphatidylinositols; Phospholipase C delta; Transient receptor potential channels

Figure

• 

  Fig. 1 TRPC4β and PLCδ1 colocalize together. (A, C) Localizations of TRPC channel and PLCδ enzyme. Upper panel: Channel with PLCδ1; Lower panel: Channel with PLCδ3. ECFP-tagged channel and EYFP-tagged PLCδ were co-expressed in HEK293 cells. The line scanned position is indicated by arrow in overlay images. Line scan graph shows TRPC4β colocalized with PLCδ1, but not with PLCδ3. Original magnification, ×63. (B, D) Summaries of FRET efficiency in the same expression conditions. The numbers in parentheses refer to cell numbers. (E) Representative blots of Co-IP experiments. HEK293 cells were co-expressed with Flag-tagged channel and EYFP-tagged PLCδ. Proteins from each condition were subjected to immunoprecipitation using anti-GFP antibody and probed with anti-Flag antibody. TRPC4β interacts with PLCδ1 directly but not with another subtype. TRPC5 interacts with neither. Data are expressed as mean ± SEM. TRPC, transient receptor potential canonical; PLC, phospholipase C; Co-IP, Co-immunoprecipitation.

• 

  Fig. 2 PLCδ1 inhibits TRPC4β currents. (A) Rapamycin-induced translocation of CFP-FKBP-PLCδ1 to the plasma membrane. CFP-FKBP-PLCδ1 and Lyn-FRB were co-expressed in HEK293 cells, and 50 nM rapamycin was used. The line scanned region is indicated by dashed line. Scale bars, 10 μm. (B, C) Representative whole-cell current recordings of HEK293 cells co-expressed with TRPC4β, FKBP-PLCδ1 in the absence (B) or presence of Lyn-FRB (C). Left panel: Time course of currents at ±100 mV every 10 sec; Right panel: I-V relationship for selected time points. Stippled lines indicate zero currents. Applications of 50 nM rapamycin and 100 nM (-)-Englerin A (EA) are indicated. The pipette solution contained 100 nM free Ca2+. (D) Summaries of peak current densities at –60 mV induced by rapamycin and EA. Rapamycin-induced PLCδ1 translocation to plasma membrane significantly reduced TRPC4β currents. Data are expressed as mean ± SEM. TRPC, transient receptor potential canonical; PLC, phospholipase C. *p < 0.05 by t-test. The numbers in parentheses refer to cell numbers.

• 

  Fig. 3 Ca2+-dependent activation of PLCδ1 occurs in physiological intracellular calcium range. (A, C, E) Representative whole-cell current recordings of HEK293 cells co-expressed with TRPC4β in the absence or presence of PLCδ1 using 50 nM (A), 100 nM (C), and 500 nM (E) free Ca2+ recording pipette solutions. Left panel: Time course of currents at ±100 mV every 10 sec; Left panel: I-V relationship for selected time points. Stippled lines indicate zero currents. Applications of 100 nM (-)-Englerin A (EA) are indicated. (B, D, F) Summaries of peak current densities at –60 mV induced by EA. The PLCδ1 inhibited TRPC4 currents when using 100 nM and 500 nM [Ca2+]i recording solutions. Data are expressed as mean ± SEM. TRPC, transient receptor potential canonical; PLC, phospholipase C. **p < 0.01, ***p < 0.001 by t-test. The numbers in parentheses refer to cell numbers.

• 

  Fig. 4 Channel calcium-activated PLCδ1 accelerates PIP2 depletion. (A, B) PIP2 depletion from plasma membrane to cytosol after (-)-Englerin A stimulation. Imaging was performed in the cells expressing TRPC4β and CFP-PH domain in the absence (A) or presence of PLCδ1 (B). PIP2 changes are monitored by CFP-PH domain. Left panel: Images of CFP-PH domain for selected time points; Right panel: Line scan graph of each image. The line scanned regions are indicated by dashed line. Scale bars, 10 μm. TRPC, transient receptor potential canonical; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate.

• 

  Fig. 5 Nonfunctional PLCδ1 mutants on PIP2 level have no effect on TRPC4 currents. (A) Representative whole cell current recordings of HEK293 cells co-expressed with TRPC4β and PLCδ1 (K30A/K32A) (A) or PLCδ1 (H311A) (B). Left panel: Time course of currents at ±100 mV every 10 sec; Right panel: I-V relationship for selected time points. Stippled lines indicate zero currents. Application of 100 nM (-)-Englerin A (EA) are indicated. The pipette solution contained 100 nM free Ca2+. (C) Schematization of PLCδ1. The mutation sites are indicated with arrow. (D) Summaries of peak current densities at –60 mV induced by EA. PLCδ1 mutants had no effect on TRPC4β currents as the PLCδ1 non-expressing cells. Data are expressed as mean ± SEM. TRPC, transient receptor potential canonical; PLC, phospholipase C. **p < 0.01, ***p < 0.001 by t-test. The numbers in parentheses refer to cell numbers.

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