Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

Abstract

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.

Keywords: G proteins; TRP channels; calcium; pertussis toxin; phospholipase C.

Fig. 1.

Differential effects of G_q/11 and G_i/o stimulation on TRPC4 activation. (A–C) Representative whole-cell currents of HEK293 cells co-expressing µOR and TRPC4β (µOR/C4β cells). The pipette solution contained 0.2 mM EGTA and no Ca²⁺. Currents were continuously recorded at –60 mV; voltage ramps from +100 to –100 mV (each held for 100 ms) within 500 ms were applied every 10 s (shaded box), which gave the vertical lines in current traces and are expanded to show the I–V relationship for selected time points (Right sides). Dashed lines indicate zero current. CCh (10 µM) and/or DAMGO (1 µM) was added as indicated. The I–V curves before and after CCh application in A are expanded to show the very weak effect of CCh on TRPC4 activation. For DAMGO stimulation alone (B), cells were divided into group I (Grp I, B₁) and group II (Grp II, B₂) as DAMGO-responsive and irresponsive, respectively. Co-application of DAMGO and CCh evoked currents in all cells (C). (D) Peak current density at –60 mV induced by DAMGO and/or CCh for individual cells. Blue boxes and bars show means ± SEM. Note the clear segregation between Grp I and Grp II cells in response to DAMGO alone was abolished by CCh. (E) Summary of T₉₀ at –60 mV for cells stimulated with DAMGO and/or CCh. **P < 0.01, ***P < 0.001, ****P < 0.0001 by t test. Cell numbers are shown in parentheses.

Fig. 2.

Dual effects of PIP₂ on G_i/o-mediated TRPC4 activation. µOR/C4β cells were transiently transfected with either a control vector or cDNA encoding DrVSP. The pipette solution had 0.05 mM EGTA and no Ca²⁺. (A) Cells were held at –60 mV, whereas depolarization pulses (100 mV, 0.5 s) were applied every 5 s. DAMGO (1 µM) and CCh (10 µM) were applied as indicated. Note the decrease in current amplitude immediately following each pulse in the DrVSP-transfected cell. Sections pointed by the arrowheads are expanded on right with I₀ (current before pulse) and I (current after pulse) indicated. (B) Time courses of I/I_o for control and DrVSP cells (means ± SEM, n = 6 for each). No current depression was detected in control cells or before DAMGO in DrVSP cells. (C) Summary of T₅₀ and T₉₀ by DAMGO. Although T₉₀ did not reach statistical significance, T₅₀ values are different. The near doubling of T₉₀ versus T₅₀ in DrVSP cells indicates monophasic current development. (D) Subthreshold DAMGO (30 nM) activated TRPC4β currents in DrVSP but not control cells. Depolarization pulses were applied at 10-s intervals. (E) Summary of peak current density at –60 mV for experiments illustrated in D. *P < 0.05 versus controls by t test.

Fig. 3.

Intracellular Ca²⁺ improves the probability but not the rate of G_i/o-mediated TRPC4 activation. µOR/C4β cells were used. (A–C) Heparin suppressed TRPC4 activation by DAMGO ± CCh. Vehicle (Veh, A) or heparin (3 mg/mL, B) was infused into cells by pipette dialysis for >5 min before DAMGO (1 µM), CCh (10 µM), and IM (10 µM) were applied extracellularly. Whole-cell currents and I–V curves show a lack of or very weak response to DAMGO and DAMGO + CCh in a heparin-treated cell. IM rescued the response (B). (C) Peak current density at –60 mV induced by DAMGO ± CCh or IM for individual cells treated or not by heparin. Blue boxes and bars show means ± SEM. (D–F) Facilitation of TRPC4 activation by IM. DAMGO-irresponsive cells (Grp II) became activated in the presence of IM (D), but IM ± CCh failed to elicit a current until DAMGO was introduced (E). (F) Peak current density at –60 mV induced by DAMGO, CCh, and IM. Only Grp II cells were further treated with IM. (G) T₉₀ for currents evoked by DAMGO + IM ± CCh. The pipette solution contained 0.2 mM EGTA and no Ca²⁺. **P ≤ 0.01, ***P ≤ 0.001 by ANOVA for C; t test for F and G.

Fig. 4.

PLCδ1 is necessary for DAMGO-evoked TRPC4 activation. µOR/C4β cells were transiently transfected with either a control vector (GFP) or cDNA encoding various PLC constructs as shown. The pipette solution contained 0.05 mM EGTA and no Ca²⁺. (A) Representative whole-cell currents in response to DAMGO (1 µM) and CCh (10 µM). DN, dominant-negative, E341R/D343R for PLCδ1 and E382R/D384R for PLCδ3; WT, wild type. Note the lack of current with PLCδ1-DN and the faster rate of DAMGO-evoked current with PLCδ1-WT and PLCδ3-WT compared with PLCβ1/β2. (B) Summary of peak current density at –60 mV evoked by DAMGO ± CCh. (C) T₉₀ for DAMGO-evoked current at –60 mV. ***P < 0.001 for δ1-DN versus GFP/δ1-WT/δ3-WT/δ3-DN/β1-WT/β2-WT and δ1-H311A versus GFP/δ1-WT/δ3-WT/β1-WT in B and δ1-WT/δ3-WT versus GFP in C; ^††P < 0.01 for δ1-H311A versus δ3-DN/β2-WT in B by ANOVA.

Fig. 5.

PLCδ1 is necessary for G_i/o activation of endogenous TRPC4-like currents in A-498 cells. (A–C) A-498 cells were transiently transfected with GFP (control) or PLCδ1 and RhoA mutants as shown. The pipette solution contained no EGTA or Ca²⁺. Whole-cell currents were recorded by voltage ramps from +100 to –100 mV (see SI Appendix, Fig. S13C) at 1 Hz. Na⁺ in the bath was replaced by Cs⁺ to facilitate current activation by adenosine (1 µM). (A) Representative time courses of currents at –75 and +75 mV (Left) and I–V curves (Right) at the indicated time points. (B) Peak current density at +75 mV evoked by adenosine for individual cells and means ± SEM (blue boxes and bars). (C) T₉₀ for adenosine-evoked currents at +75 mV. *P < 0.05 versus GFP by t test. (D) Diagram of PLCδ1–TRPC4 self-reinforcing system. Positive and negative effects necessary for TRPC4 activation are indicated in green and red lines, respectively. Nonessential, modulatory effects are indicated in black. In addition to activating PLCδ1, Ca²⁺ might directly enhance channel function, based on previous work with TRPC5 and native channels containing TRPC4. Thus, at least three self-reinforcing loops exist to sustain TRPC4 activity when G_i/o is active: positive feedback effects between Ca²⁺ and TRPC4 (i); among PLCδ1, Ca²⁺ and TRPC4 (ii); as well as a double-negative effect among PLCδ1, PIP₂, and TRPC4, which is continuously supported by Ca²⁺ that links TRPC4 function to PLCδ1 activation (iii). The system is facilitated by the G_q/11–PLCβ pathway, which provides the Ca²⁺ signal for PLCδ1 activation and helps lower the barrier of channel gating by reducing PIP₂. It is negatively regulated by RhoA (through inhibiting PLCδ1) and high Ca²⁺ levels, which may inhibit either TRPC4 or PLCδ1, or both (dashed lines).