Zheng W et al. (2018), Direct Binding between Pre-S1 and TRP-like Doma...

XB-ART-55973

Cell Rep 2018 Feb 06;226:1560-1573. doi: 10.1016/j.celrep.2018.01.042.

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Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2.

Zheng W , Cai R , Hofmann L , Nesin V , Hu Q , Long W , Fatehi M , Liu X , Hussein S , Kong T , Li J , Light PE , Tang J , Flockerzi V , Tsiokas L , Chen XZ .

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Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels.

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Species referenced: Xenopus laevis
Genes referenced: ctrl foxm1 pkd1 pkd2 trpm8 trpv1 wnt9b

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	Figure 2. Roles of the TRPP2 Residues W201 (Pre-S1 Domain) and K688 (TRP-like Domain) in the Channel Function and N-C Binding(A) Sequence alignment of pre-S1 and TRP-like helices among human TRPP channels.(B) Left panel: representative current-voltage (I-V) curves obtained from oocytes expressing human WT or a mutant TRPP2, as indicated, in the presence of a Na+-containing, divalent-free solution (in mM): 100 NaCl; 2 KCl; and 10 HEPES (pH 7.5). Right panel: averaged currents at +80 mV are shown. Ctrl, H2O-injected oocytes. Currents were averaged from 13â€“17 oocytes of three batches. *p < 0.001.(C) Representative immunoblots of surface biotinylated and total proteins of TRPP2 WT and mutants.(D) Left panel: representative co-IP data showing the effects of TRPP2 W201 and K688 on the N-C interaction using oocytes co-expressing HA-tagged human FL TRPP2 and Flag-tagged TRPP2 N-terminal peptide (Flag-P2NP; G161-S215). Right panel: data from experiments in left panel were quantified, averaged, and normalized. p < 0.01; n = 3.(E) Blocking effects of Flag-P2NP and Flag-P2NP-W201A on channel function of FL TRPP2 mutant F604P by use of co-expression. None, no P2NP was co-expressed with FL F604P. Normalized currents at +80 mV were obtained and averaged from three independent experiments. ***p < 0.001.Data are presented as mean Â± SEM. See also Figures S2 and S6.
	Figure 3. Roles of TRPP2 Residues W201 and K688 in WNT9B-Induced Whole-Cell Currents(A) Cell surface expression of WT TRPP2 and mutants W201A, Î›Î¥684, Y684A, and K688A in HEK293 cells by transient co-transfection together with PKD1.(B) Representative whole-cell current traces obtained at +100 and âˆ’100 mV before and after extracellular addition of WNT9B (0.5 Î¼g/mL) or La3+ (100 Î¼M) in CHO-K1 cells transiently co-expressing PKD1 with WT TRPP2, the W201A, or K688A mutant. The electrophysiological measurements were performed as described previously (Kim et al., 2016).(C) Averaged steady-state I-V curves obtained before and ~2 min after application of WNT9B, at time points indicated by arrows in (B). WT TRPP2, n = 7 cells; W201A, n = 11 cells; K688A, n = 7 cells.(D and E) Averaged peak (D) and steady-state (E) currents induced by WNT9B in CHO-K1 cells under the same experimental conditions as in (B). WT TRPP2, n = 7 cells; W201A, n = 11 cells; Î›Y684, n = 8 cells; Y682A, n = 10 cells; K688A, n = 7 cells. p < 0.05; p < 0.01; **p < 0.001.Data are presented as mean Â± SEM.
	Figure 4. Roles of TRPM8 and TRPV1 Aromatic and Cationic Residues in the Pre-S1 and TRP-like Domains, Respectively, in the N-C Binding and Channel Function(A) Sequence alignment of human TRPMs pre-S1 and TRP-like domains.(B) Left panel: representative whole-cell I-V curves obtained from oocytes expressing rat TRPM8 WT or a mutant channel in the presence of Na+-containing extracellular solution at RT. Ctrl, H2O-injected oocytes. Right panel: averaged currents at +80 mV from 12â€“18 oocytes of three batches are shown.(C) Left panel: representative current traces obtained at âˆ’50 mV from oocytes expressing WT or a mutant TRPM8 before and after addition of 0.5 mM menthol. Right panel: averaged menthol-induced currents from 12â€“18 oocytes of three batches are shown.(D) Ca2+-imaging measurements showing averaged fura-2 ratios obtained before and after Ca2+ (2 mM) and menthol (0.5 mM) addition to Na+-containing extracellular solution in HEK293 cells transiently co-expressing GFP with rat WT or a mutant TRPM8 or none (Ctrl) at 37Â°C.(E and F) Representative co-IP data using oocytes expression, showing the interaction of Flag-tagged FL TRPM8 with HA-tagged TRPM8 N-terminal peptide (HA-M8NP; N642-K691; E) or C-terminal peptide (HA-M8CP; G980-F1029; F).(G) Left panel: representative whole-cell I-V curves obtained from oocytes expressing rat WT or a mutant TRPV1 in the presence of the Na+-containing solution at RT. Ctrl, H2O-injected oocytes. Right panel: averaged currents at +120 mV from 10â€“16 oocytes of three batches are shown.(H) Left panel: representative current traces obtained at â€”50 mV in rat WT or a mutant TRPV1 expressing oocytes before and after extracellular addition of capsaicin (15 Î¼M). Right panel: averaged capsaicin-induced currents from 10â€“16 oocytes of three batches are shown.(I and J) Representative co-IP data using oocytes expression, showing the interaction of rat FL TRPV1 with Flag-tagged TRPV1 N-terminal peptide (Flag-V1NP; D383-R432; I) or C-terminal peptide (Flag-V1CP; N687-D736; J).Data are presented as mean Â± SEM. See also Figures S3, S4, S5, and S6.
	Figure 5. Inhibition of the TRPP3 Channel Function and N-C Interaction by PIP2(A) Averaged and normalized Ca-activated currents obtained from TRPP3-expressing oocytes before and after on-site injection of 25 nL water without (Ctrl) or containing diC8 (5 mM). Injection was performed with a third electrode after the initial current measurement. The second current measurement was performed 10 min after the injection. Currents were averaged from three independent experiments (with n = 12â€“15). *p < 0.001.(B) Averaged and normalized Ca-activated currents obtained from TRPP3-expressing oocytes pre-incubated with 10 Î¼M wortmannin or DMSO (Ctrl) for 1 hr before measurements. p < 0.01.(C) Alignment of the C-terminal putative PIP2 binding domains in human TRPPs. Conserved cationic residues are highlighted.(D) Averaged Ca-activated currents obtained from oocytes expressing WT or a mutant TRPP3 (n = 14â€“18). p < 0.05; p < 0.01; and p < 0.001.(E) Averaged and normalized Ca-activated currents. QQRK, R594Q/R596Q double mutant; QQQK, R594Q/R596Q/R598Q triple mutant; QQQQ, R594Q/R596Q/ R598Q/K599Q quadruple mutant. p < 0.01 and *p < 0.001.(F) Left panel: representative co-IP data showing interaction between diC8 PIP2 and TRPP3. Right panel: representative co-IP data show interaction of diC8 PIP2 with WT or a mutant TRPP3 expressed in oocytes. DR594-K599, TRPP3 deleted with fragment R594-K599. diC8 PIP2 or phosphatidic acid (PA, a negative control) was added to cell lysate to a final concentration of 15 Î¼M. An anti-PIP2 antibody (sc-53412) from Santa Cruz Biotechnology was used for immuno-precipitation.(G) Averaged and normalized Ca-activated currents obtained from oocytes expressing WT TRPP3 or the QQQQ mutant. Oocytes were treated with 10 Î¼M wortmannin or DMSO (Ctrl) for 1 hr before measurements. p < 0.01; NS, not significant.(H) Left panel: representative co-IP data showing the effect of diC8 on the interaction of P3NP with FL TRPP3 in oocytes. diC8 was added in the cell lysis buffer to final concentration of 15 Î¼M. Right panel: data from three independent experiments in left panel were quantified, averaged, and normalized. ***p < 0.001.(I) Left panel: representative co-IP data showing the effect of diC8 on the interaction of P3NP with the TRPP3 QQQQ mutant. Right panel: data from three independent experiments in left panel were quantified, averaged, and normalized (NS, not significant).(J) Left panel: representative co-IP data showing the effect of diC8 on the interaction of P2NP with FL TRPP2. Right panel: data from three independent experiments in left panel were quantified, averaged, and normalized.(K) Representative co-IP data showing the interaction of PIP2 with expressed WT or a mutant TRPP3 in oocytes.Data are presented as mean Â± SEM. See also Figure S2.
	Figure 6. Effects of PIP2 on the N-C Interaction in TRPM8 and TRPV1(A and B) Left panels: representative co-IP data showing the effect of diC8 on the interaction between FL TRPM8 and M8NP (A) or M8CP (B) under the same experimental conditions as in Figure 4H. Right panels: data from three independent experiments in left panel were quantified, averaged, and normalized. p < 0.05.(C and D) Left panels: representative co-IP data showing the effect of diC8 on the interaction between the FLTRPV1 and V1NP (C) or V1CP (D). Right panels: data from three independent experiments in left panel were quantified, averaged, and normalized. *p < 0.01.(E) Schematic model showing role of the PIP2-regulated, W-K/R pair-mediated N-C binding in TRP channels activation and function. In this model, the N-C binding is mediated between the conserved aromatic residue Win pre-S1 and the cationic residue K or Rin the TRP/TRP-like domain and is either inhibited (as in TRPP3 and -P2) or enhanced (as in TRPM8 and -V1) by PIP2 through binding to the TRPs protein. The presence of the N-C binding is required for TRPs agonists to activate the channel.Data are presented as mean Â± SEM.

References [+] :

Arif Pavel, Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant. 2016, Pubmed

	Figure 2. Roles of the TRPP2 Residues W201 (Pre-S1 Domain) and K688 (TRP-like Domain) in the Channel Function and N-C Binding(A) Sequence alignment of pre-S1 and TRP-like helices among human TRPP channels.(B) Left panel: representative current-voltage (I-V) curves obtained from oocytes expressing human WT or a mutant TRPP2, as indicated, in the presence of a Na+-containing, divalent-free solution (in mM): 100 NaCl; 2 KCl; and 10 HEPES (pH 7.5). Right panel: averaged currents at +80 mV are shown. Ctrl, H2O-injected oocytes. Currents were averaged from 13â€“17 oocytes of three batches. *p < 0.001.(C) Representative immunoblots of surface biotinylated and total proteins of TRPP2 WT and mutants.(D) Left panel: representative co-IP data showing the effects of TRPP2 W201 and K688 on the N-C interaction using oocytes co-expressing HA-tagged human FL TRPP2 and Flag-tagged TRPP2 N-terminal peptide (Flag-P2NP; G161-S215). Right panel: data from experiments in left panel were quantified, averaged, and normalized. p < 0.01; n = 3.(E) Blocking effects of Flag-P2NP and Flag-P2NP-W201A on channel function of FL TRPP2 mutant F604P by use of co-expression. None, no P2NP was co-expressed with FL F604P. Normalized currents at +80 mV were obtained and averaged from three independent experiments. ***p < 0.001.Data are presented as mean Â± SEM. See also Figures S2 and S6.
	Figure 3. Roles of TRPP2 Residues W201 and K688 in WNT9B-Induced Whole-Cell Currents(A) Cell surface expression of WT TRPP2 and mutants W201A, Î›Î¥684, Y684A, and K688A in HEK293 cells by transient co-transfection together with PKD1.(B) Representative whole-cell current traces obtained at +100 and âˆ’100 mV before and after extracellular addition of WNT9B (0.5 Î¼g/mL) or La3+ (100 Î¼M) in CHO-K1 cells transiently co-expressing PKD1 with WT TRPP2, the W201A, or K688A mutant. The electrophysiological measurements were performed as described previously (Kim et al., 2016).(C) Averaged steady-state I-V curves obtained before and ~2 min after application of WNT9B, at time points indicated by arrows in (B). WT TRPP2, n = 7 cells; W201A, n = 11 cells; K688A, n = 7 cells.(D and E) Averaged peak (D) and steady-state (E) currents induced by WNT9B in CHO-K1 cells under the same experimental conditions as in (B). WT TRPP2, n = 7 cells; W201A, n = 11 cells; Î›Y684, n = 8 cells; Y682A, n = 10 cells; K688A, n = 7 cells. p < 0.05; p < 0.01; **p < 0.001.Data are presented as mean Â± SEM.
	Figure 4. Roles of TRPM8 and TRPV1 Aromatic and Cationic Residues in the Pre-S1 and TRP-like Domains, Respectively, in the N-C Binding and Channel Function(A) Sequence alignment of human TRPMs pre-S1 and TRP-like domains.(B) Left panel: representative whole-cell I-V curves obtained from oocytes expressing rat TRPM8 WT or a mutant channel in the presence of Na+-containing extracellular solution at RT. Ctrl, H2O-injected oocytes. Right panel: averaged currents at +80 mV from 12â€“18 oocytes of three batches are shown.(C) Left panel: representative current traces obtained at âˆ’50 mV from oocytes expressing WT or a mutant TRPM8 before and after addition of 0.5 mM menthol. Right panel: averaged menthol-induced currents from 12â€“18 oocytes of three batches are shown.(D) Ca2+-imaging measurements showing averaged fura-2 ratios obtained before and after Ca2+ (2 mM) and menthol (0.5 mM) addition to Na+-containing extracellular solution in HEK293 cells transiently co-expressing GFP with rat WT or a mutant TRPM8 or none (Ctrl) at 37Â°C.(E and F) Representative co-IP data using oocytes expression, showing the interaction of Flag-tagged FL TRPM8 with HA-tagged TRPM8 N-terminal peptide (HA-M8NP; N642-K691; E) or C-terminal peptide (HA-M8CP; G980-F1029; F).(G) Left panel: representative whole-cell I-V curves obtained from oocytes expressing rat WT or a mutant TRPV1 in the presence of the Na+-containing solution at RT. Ctrl, H2O-injected oocytes. Right panel: averaged currents at +120 mV from 10â€“16 oocytes of three batches are shown.(H) Left panel: representative current traces obtained at â€”50 mV in rat WT or a mutant TRPV1 expressing oocytes before and after extracellular addition of capsaicin (15 Î¼M). Right panel: averaged capsaicin-induced currents from 10â€“16 oocytes of three batches are shown.(I and J) Representative co-IP data using oocytes expression, showing the interaction of rat FL TRPV1 with Flag-tagged TRPV1 N-terminal peptide (Flag-V1NP; D383-R432; I) or C-terminal peptide (Flag-V1CP; N687-D736; J).Data are presented as mean Â± SEM. See also Figures S3, S4, S5, and S6.
	Figure 5. Inhibition of the TRPP3 Channel Function and N-C Interaction by PIP2(A) Averaged and normalized Ca-activated currents obtained from TRPP3-expressing oocytes before and after on-site injection of 25 nL water without (Ctrl) or containing diC8 (5 mM). Injection was performed with a third electrode after the initial current measurement. The second current measurement was performed 10 min after the injection. Currents were averaged from three independent experiments (with n = 12â€“15). *p < 0.001.(B) Averaged and normalized Ca-activated currents obtained from TRPP3-expressing oocytes pre-incubated with 10 Î¼M wortmannin or DMSO (Ctrl) for 1 hr before measurements. p < 0.01.(C) Alignment of the C-terminal putative PIP2 binding domains in human TRPPs. Conserved cationic residues are highlighted.(D) Averaged Ca-activated currents obtained from oocytes expressing WT or a mutant TRPP3 (n = 14â€“18). p < 0.05; p < 0.01; and p < 0.001.(E) Averaged and normalized Ca-activated currents. QQRK, R594Q/R596Q double mutant; QQQK, R594Q/R596Q/R598Q triple mutant; QQQQ, R594Q/R596Q/ R598Q/K599Q quadruple mutant. p < 0.01 and *p < 0.001.(F) Left panel: representative co-IP data showing interaction between diC8 PIP2 and TRPP3. Right panel: representative co-IP data show interaction of diC8 PIP2 with WT or a mutant TRPP3 expressed in oocytes. DR594-K599, TRPP3 deleted with fragment R594-K599. diC8 PIP2 or phosphatidic acid (PA, a negative control) was added to cell lysate to a final concentration of 15 Î¼M. An anti-PIP2 antibody (sc-53412) from Santa Cruz Biotechnology was used for immuno-precipitation.(G) Averaged and normalized Ca-activated currents obtained from oocytes expressing WT TRPP3 or the QQQQ mutant. Oocytes were treated with 10 Î¼M wortmannin or DMSO (Ctrl) for 1 hr before measurements. p < 0.01; NS, not significant.(H) Left panel: representative co-IP data showing the effect of diC8 on the interaction of P3NP with FL TRPP3 in oocytes. diC8 was added in the cell lysis buffer to final concentration of 15 Î¼M. Right panel: data from three independent experiments in left panel were quantified, averaged, and normalized. ***p < 0.001.(I) Left panel: representative co-IP data showing the effect of diC8 on the interaction of P3NP with the TRPP3 QQQQ mutant. Right panel: data from three independent experiments in left panel were quantified, averaged, and normalized (NS, not significant).(J) Left panel: representative co-IP data showing the effect of diC8 on the interaction of P2NP with FL TRPP2. Right panel: data from three independent experiments in left panel were quantified, averaged, and normalized.(K) Representative co-IP data showing the interaction of PIP2 with expressed WT or a mutant TRPP3 in oocytes.Data are presented as mean Â± SEM. See also Figure S2.
	Figure 6. Effects of PIP2 on the N-C Interaction in TRPM8 and TRPV1(A and B) Left panels: representative co-IP data showing the effect of diC8 on the interaction between FL TRPM8 and M8NP (A) or M8CP (B) under the same experimental conditions as in Figure 4H. Right panels: data from three independent experiments in left panel were quantified, averaged, and normalized. p < 0.05.(C and D) Left panels: representative co-IP data showing the effect of diC8 on the interaction between the FLTRPV1 and V1NP (C) or V1CP (D). Right panels: data from three independent experiments in left panel were quantified, averaged, and normalized. *p < 0.01.(E) Schematic model showing role of the PIP2-regulated, W-K/R pair-mediated N-C binding in TRP channels activation and function. In this model, the N-C binding is mediated between the conserved aromatic residue Win pre-S1 and the cationic residue K or Rin the TRP/TRP-like domain and is either inhibited (as in TRPP3 and -P2) or enhanced (as in TRPM8 and -V1) by PIP2 through binding to the TRPs protein. The presence of the N-C binding is required for TRPs agonists to activate the channel.Data are presented as mean Â± SEM.