Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels

Abstract

Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gα_q-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) maintains TRPC4 and TRPC5 channel function, the Gα_q-PLC pathway inhibits channel activity by depleting PI(4,5)P₂. Here we investigated the difference in PI(4,5)P₂ sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P₂ dephosphorylation robustly inhibits TRPC4α, TRPC4β, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4β, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P₂ dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4β < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P₂ binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P₂ binding sites because the mutations reduced FRET to a PI(4,5)P₂ sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P₂ affinity for TRPC4 and TRPC5 that changes PI(4,5)P₂ sensitivity.

Figure 1

DrVSP mediated PH probe FRET reduction and current inhibition. (a) Principles of measurement of PI(4,5)P₂ sensor FRET in cells transfected with PI(4,5)P₂ sensor (PLCδ1 PH-domain) fused to CFP or YFP and DrVSP. (b) Pseudocolor FRET images of PI(4,5)P₂ sensor during progressive depolarization from +10 to +140 mV in 10 mV steps. (c,d) FRET efficiency change caused by step-pulse protocol (from +10 to +140 mV; duration of 500 ms; repeated every 30 s) from cells transfected with CFP-PH, YFP-PH and DrVSP (c) or DrVSP_C302S (d). The area enclosed by the dashed box includes enlarged form (right). (e,f) Current change caused by step-pulse protocol from cells transfected with TRPC4α and DrVSP (e) or DrVSP_C302S (f). Englerin A (EA, 100 nM) was applied to induce the currents. The area enclosed by the dashed box includes enlarged form (right).

Figure 2

DrVSP-mediated current inhibition of TRPC4, TRPC5 homotetramers and TRPC1/4, TRPC1/5 heterotetramers. (a,b) Gradual current inhibition in TRPC4α, TRPC4β and TRPC5 homotetramers (a) and TRPC1/4α, TRPC1/4β and TRPC1/5 heterotetramers (b). The conditions are largely same as in Fig. 1e except transfected channels. (c,d) Current inhibition for different voltages in homotetramers (c) and heterotetramers (d).

Figure 3

Quantification of PI(4,5)P₂ dissociation from binding to homo- and heteromeric channels. (a–f) Ratio of voltage dependent current inhibition and FRET reduction after DrVSP activation in cells expressing TRPC4α (n = 6) (a), TRPC4β (n = 7) (b) and TRPC5 (n = 10) (c) homotetramers and TRPC1/4α (n = 4) (d), TRPC1/4β (n = 8) (e) and TRPC1/5 (n = 7) (f) heterotetramers. (g) r(I) against estimated PI(4,5)P₂ concentration plots based on the conversion from FRET to PI(4,5)P₂ of homotetramers. (h) Hill plots for homotetramers, enclosed by the dashed box is at a higher PI(4,5)P₂ concentration resolution. (i,j) The conditions are largely same as in Fig. 3g,h, except heterotetramers instead.

Figure 4

Molecular model of TRPC4 based on the structure of NOMPC and TRPM4. (a) Schematization of a TRPC4 subunit. (b–f) Expended views of putative PI(4,5)P₂ binding regions in prediction model of TRPC4.

Figure 5

Putative PI(4,5)P₂ binding sites of TRPC4 and its functional change. (a) Summary of FRET efficiency of TRPC4 mutants. (b) Images of FRET between CFP tagged channels (wild-type TRPC4β and mutants K419, K518A and K664A) and YFP-PH. (c) Current potentiation of wild-type TRPC4 or mutant channels transfected with Gα_i2 (Q209L). The I-V relationships of wild-type and mutants which shows significantly reduced currents (right). (d) EA (100 nM) stimulation of wild-type TRPC4 or mutant. The I-V relationships of wild-type and mutants (right). (e) Basal current density of mutants which showed decreased activities comparison with wild-type TRPC4β. (f,g) Current inhibition for different voltages (f) or different time durations (g) in WT, R511A, H630A (left) and K419A, K518A, K664A (right). Data are presented as mean ± SEM and analyzed using student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.