Korean J Physiol Pharmacol.  2019 Sep;23(5):357-366. 10.4196/kjpp.2019.23.5.357.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

• Ko J
• Myeong J
• Kwak M
• Jeon JH
• So I

• KMID: 2455812
• DOI: http://doi.org/10.4196/kjpp.2019.23.5.357

Abstract

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)Pâ‚‚) depletion. When PI(4,5)Pâ‚‚ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²âº release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²âº due to Ca²âº release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)Pâ‚‚ depletion.

Keyword

Calcium; GTP-binding proteins; Protein kinase C; Transient receptor potential channels; Type C phospholipases

MeSH Terms

Calcium
Cytoplasm
Endoplasmic Reticulum
GTP-Binding Proteins
HEK293 Cells
Inositol
Phosphatidylinositol 4,5-Diphosphate
Phospholipases*
Phosphotransferases
Protein Kinase C
Transient Receptor Potential Channels
Type C Phospholipases*
Calcium
GTP-Binding Proteins
Inositol
Phosphatidylinositol 4,5-Diphosphate
Phospholipases
Phosphotransferases
Protein Kinase C
Transient Receptor Potential Channels
Type C Phospholipases

Figure

• 

  Fig. 1 Transient receptor potential canonical (TRPC) 1α/4β, TRPC1α/5 currents inhibition depend on cytosolic calcium level. (A) Catalytic scheme for phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) by Danio rerio voltage-sensitive lipid phosphatase (DrVSP), inositol polyphosphate 5-phosphatase (Inp54) (left) and, the phospholipase C beta (PLCβ) (right) which produces PI(4)P/Pi and diacylglycerol (DAG)/inositol trisphosphate (IP3), respectively. (B, C) Representative currents trace of TRPC1α/4β (B) and TRPC1α/5 (C) stimulated by TG (1 µM). (D) Summary of current densities between basal and thapsigargin (TG) evoked peak in cells co-expressing YFP-TRPC1α with TRPC4β-CFP or CFP-TRPC5. (E–H) Averaged plots of currents at +100 mV every 10 sec upon carbachol (CCh, 100 µM) stimulation from cells co-expressing muscarinic receptor 3 (M3R), YFP-TRPC1α, and TRPC4β-CFP (E, F) or CFP-TRPC5 (G, H). (F) and (H) show the normalized (norm.) currents with internal solution that contains 0 and 5 mM of 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The pipette solution contained 0, 0.5, and 5 mM of BAPTA, and each trace was normalized; TRPC1/4β (0 mM, n = 4; 0.5 mM, n = 4; 5 mM, n = 4), TRPC1/5 (0 mM, n = 4; 0.5 mM, n = 5; 5 mM, n = 4). Data are presented as mean ± standard error of the mean; n, sample number; n.s., not significant.

• 

  Fig. 2 Effects of calcium on heterotetrameric channel currents. (A–D) Rapamycin (rapa) (20 nM, 3) and subsequent addition of thapsigargin (1 µM, 4) or ionomycin (1 µM, 4) induced current inactivation in transient receptor potential canonical (TRPC) 1α/4β after Englerin A (EA) (100 nM, 2) stimulation. (A, C) Representative whole-cell currents of HEK293 cells co-expressing TRPC1α/4β, inositol polyphosphate 5-phosphatase (Inp54), Lyn11 (left) and I–V relationship for selected time points of each stimulation (right). (B, D) Summary of current density at +100 mV induced by the stimulations (thapsigargin [TG], n = 5; ionomycin [Iono], n = 4). (E–H) The conditions are largely the same as those in (A–D), but in cells expressing TRPC1α/5, Inp54, and Lyn11 (TG, n = 4; Iono, n = 5). Data are presented as mean ± standard error of the mean; n, sample number; n.s., not significant. *p < 0.05, **p < 0.01.

• 

  Fig. 3 Involvement of protein kinase C (PKC) on heteromeric channel current inhibition. (A, B) Effect of a PKC inhibitor on transient receptor potential canonical (TRPC) 1α/4β (A) and TRPC1α/5 (B) current desensitization. CFP-tagged TRPC4β or TRPC5 was coexpressed with YFP-TRPC1α, muscarinic receptor 3 (M3R). Channels were stimulated by carbachol (CCh) (100 µM) twice, with or without GF109203X pretreatment. (C) Summary of desensitization ratio in TRPC1α/4β (2 / 1) and TRPC1α/5 (2′ / 1′) with or without PKC inhibitor, GF109203X and chelerythrine. (D) Plots of currents at +100 mV every 10 sec upon CCh (100 µM) stimulation from cells expressing TRPC1α/4β and TRPC1α/4β (T887A). (E) Comparison of the basal and peak current between wild-type and T887A mutant of TRPC1α/4β. (F) Plots of normalized (norm.) currents at +100 mV upon CCh (100 µM) stimulation from cells expressing TRPC1α/4β and TRPC1α/4β (T887A). (G–I) The conditions are largely the same as those in (D–F), but in cells expressing TRPC1α/5 and TRPC1α/5 (T972A) instead. Data are presented as mean ± standard error of the mean; n.s., not significant. *p < 0.05.

• 

  Fig. 4 Effect of 1-oleoyl-2-acetyl-sn-glycerol (OAG) on heteromeric channel current. (A, B) Rapamycin (rapa) (20 nM, 3) and subsequent addition of OAG (100 µM, 4) induced current inactivation in transient receptor potential canonical (TRPC) 1α/4β after Englerin A (EA) (100 nM, 2) stimulation. (A) Representative whole-cell currents of HEK293 cells co-expressing TRPC1α/4β, inositol polyphosphate 5-phosphatase (Inp54), Lyn11 (left) and current-voltage relationship for selected time points of each stimulation (right). (B) Summary of current density at +100 mV induced by the stimulations (n = 6). (C, D) The conditions are largely the same as those in (A and B), but in the cells expressing TRPC1α/5 instead (n = 5). Data are presented as mean ± standard error of the mean; n.s., not significant. *p < 0.05.

• 

  Fig. 5 Transient receptor potential canonical (TRPC) 1/4 and TRPC1/5 channel regulation by the Gαq-phospholipase C (PLC) pathway. (A–D) Scheme of the heteromeric TRPC1/4 and TRPC1/5 with Gαq protein coupled receptor (GPCR). Channels and GPCR expressed in plasma membrane. Schematic diagram of each proteins presented as below box (bottom). (A) In the resting state, guanosine diphosphate (GDP)-bound inactive Gαq proteins are preassembled with Gαq-coupled receptors. (B) Upon association of agonist to muscarinic receptor 3 (M3R), activated Gαq releases from receptor and directly binds to active channels (activation state). Also, as a downstream of the Gαq pathway, active PLC hydrolyzes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2). (C) Depletion of the channel-bound PI(4,5)P2 inhibits channel gating (1st inactivation state). (D) Desensitization of TRPC1/4 and TRPC1/5 channels induced by active PKC and high [Ca2+]i from endoplasmic reticulum (ER) calcium release (2nd inactivation).

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