Liu X et al. (2023), Inhibition of TRPP3 by calmodulin through Ca2+/...

XB-ART-59852

Cell Insight 2023 Apr 01;22:100088. doi: 10.1016/j.cellin.2023.100088.

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Inhibition of TRPP3 by calmodulin through Ca2+/calmodulin-dependent protein kinase II.

Liu X , Wang Y , Weng Z , Xu Q , Zhou C , Tang J , Chen XZ .

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Transient receptor potential (TRP) polycystin-3 (TRPP3) is a non-selective cation channel activated by Ca2+ and protons and is involved in regulating ciliary Ca2+ concentration, hedgehog signaling and sour tasting. The TRPP3 channel function and regulation are still not well understood. Here we investigated regulation of TRPP3 by calmodulin (CaM) by means of electrophysiology and Xenopus oocytes as an expression model. We found that TRPP3 channel function is enhanced by calmidazolium, a CaM antagonist, and inhibited by CaM through binding of the CaM N-lobe to a TRPP3 C-terminal domain not overlapped with the EF-hand. We further revealed that the TRPP3/CaM interaction promotes phosphorylation of TRPP3 at threonine 591 by Ca2+/CaM-dependent protein kinase II, which mediates the inhibition of TRPP3 by CaM.

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Species referenced: Xenopus
Genes referenced: shh
GO keywords: cation channel activity

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	Graphical abstract
	Fig. 1. Effect of CMZ on TRPP3 channel function. A. Representative whole-cell current traces obtained from a water-injected oocyte (Ctrl) and one expressing human TRPP3. Oocytes were clamped at −50 mV and perfused with the Na+-containing solution (“Na+’’) (in mM) (100 NaCl, 2 KCl, 1 MgCl2, 10 HEPES, pH 7.5), or one added with 5 mM CaCl2 (“Na+ + Ca2+’’), in the presence of 10 μM MONNA throughout. TRPP3 channel activity was measured as the Ca2+-induced peak current (“Na+ + Ca2+’’) (i.e., current at ‘‘Na+ + Ca2+’’ minus current at ‘‘Na+’’). 10 μM CMZ was applied, as indicated. B. Averaged Ca2+-induced currents obtained from Ctrl oocytes and those expressing TRPP3, under the same experimental conditions as in panel A. ∗∗∗p < 0.001. C. Representative I–V curves obtained from a Ctrl and TRPP3-expressing oocyte, using a ramp protocol (with voltages, in mV, as indicated) applied at time points indicated by vertical bars in panel A traces. D. Representative whole-cell current traces recorded by TEVC at −50 mV from a Ctrl and TRPP3-expressing oocyte. Oocytes were perfused with Na+-containing solution or one with equimolar N-methyl-D-glucamine (NMDG) replacing Na+ (NMDG). 10 μM CMZ was applied, as indicated. E. Averaged Na currents from Ctrl and TRPP3-expressing oocytes. ∗p < 0.05. Na + currents were determined as the difference between the plateau (“Na+’’) and baseline (“NMDG”) values. F. Representative I–V curves from a Ctrl and TRPP3-expressing oocyte, using the ramp protocol (as in panel C) applied at time points indicated by vertical bars in panel E traces.
	Fig. 2. Effect of CaM on TRPP3 channel function and physical interaction between TRPP3 and CaM. A. Averaged basal (“Na+’’) current and Ca2+-activated current (= current at “Na+ + Ca2+” minus current at “Na+’’) measured in Ctrl and TRPP3-expressing oocytes, with or without CaM, using TEVC from three independent experiments. Correspondingly, the Na+-containing solution with 5 mM CaCl2 (“Na+ + Ca2+’’) or without Ca2+ (“Na+’’) was used. ∗∗p < 0.01; ∗∗∗p < 0.001; by One-way ANOVA with Holm-Sidak's correction. B. Left panel: representative surface and total expression of TRPP3 and CaM determined by biotinylation, with β-actin as a loading control. Right panel: data from three independent experiments were quantified, normalized, and averaged. ns: not significant. C. Representative co-IP data of three independent experiments, showing interaction between TRPP3 and CaM. D. Representative CaM-Agarose Pull-down data of three independent experiments, showing direct TRPP3/CaM binding as a function of the Ca2+ concentration. E. Left panel: representative CaM-Agarose Pull-down data, showing the effect of CMZ on the direct TRPP3/CaM binding. Right panel: data from three independent experiments were quantified, normalized, and averaged. ∗∗∗p < 0.001.
	Fig. 3. Domains of interaction between CaM and TRPP3. A-C. Flag-TRPP3 was expressed in oocytes alone or with HA-tagged WT or a mutant CaM (CaM12, CaM34 or CaM1234). A. Averaged basal (“Na+’’) and Ca2+-activated (“Na+ + Ca2+” minus “Na+’’) currents from three independent experiments measured using TEVC in the presence of the Na-containing solution with (“Na+ + Ca2+’’) or without (“Na+’’) 5 mM CaCl2. ∗p < 0.05; ns: not significant. B. Surface and total expression of TRPP3 and CaM mutants determined by biotinylation as representative data of three independent experiments, with β-actin as a loading control. C. Representative co-IP data of three independent experiments, showing the interaction between TRPP3 and a CaM mutant. D. Representative CaM-Agarose Pull-down data of three independent experiments, showing direct interaction of CaM with WT TRPP3, P3ΔN or P3ΔC. E. Left panel: representative GST Pull-down data showing direct interaction of His-P3CP with GST-tagged CaM with purified GST and GST-P3NP as negative and positive controls, respectively. Right panel: data from three independent experiments, as in left panel, were quantified, normalized, and averaged. F. Representative co-IP data of three independent experiments, showing the interaction of HA-CaM with Flag-TRPP3 (WT or mutant T622X). G. Representative GST Pull-down data of three independent experiments, showing the interaction of GST-CaM (with GST as control) with Flag-tagged TRPP3 WT, truncation mutant T622X, or P3ΔC (Ctrl). H. Ca2+-activated currents of WT TRPP3 or mutant T622X with or without CaM co-expression in oocytes were averaged from three independent experiments in the Na+-containing solution. ∗∗∗p < 0.001; ns: not significant.
	Fig. 4. Relationship of CaM and PIP2 on TRPP3 regulation. A. Left panel: representative co-IP data showing the effect of CaM on the interaction of diC8 PIP2 (a water-soluble dioctanoyl analog of PIP2) with TRPP3, with TRPP3 4Q mutant (R594Q/R596Q/R598Q/K599Q quadruple mutant) as a control. Right panel: data from three independent experiments in left panel were quantified, normalized, and averaged. ∗∗p < 0.01. B. Left panel: representative GST Pull-down data showing the effect of CaM on the interaction between GST-P3NP and Flag-TRPP3 FL, with diC8 PIP2 as a positive control. Right panel: data from three independent experiments, as in left panel, were quantified, normalized, and averaged. ∗∗p < 0.01; ns: not significant.
	Fig. 5. Effect of CaMK2 on TRPP3 channel function. A. Ca2+-activated currents (at −50 mV) averaged from three batches of TRPP3-expressing oocytes after treatment with 10 μM KN93, 10 μM AIP or 0.1% DMSO (Ctrl) for 30 min ∗∗p < 0.01. B. Averaged Ca2+-activated currents (at −50 mV) obtained from three batches of oocytes co-expressing TRPP3 with an indicated CaMK2 mutant or none. ∗∗∗p < 0.001; ns: not significant. C. Effects of CaMK2 mutants on the TRPP3 expression on the oocyte surface. Upper panel: representative surface and total expression of TRPP3 determined by biotinylation, with β-actin as a loading control. Lower panel: data from three independent experiments were quantified, normalized, and averaged. ns: not significant. D. Representative co-IP data of three independent experiments, showing the interaction between TRPP3 and CaMK2. E. Left panel: representative co-IP data showing the effects of CaM or its mutant CaM1234 on the interaction between TRPP3 and CaMK2. Right panel: data from three independent experiments were quantified, normalized, and averaged. ∗∗∗p < 0.001, ns: not significant.
	Fig. 6. Role of T591 in the TRPP3 channel function. A. CaM-binding domain identified in the TRPP3 C-terminus in which a PIP2-binding site and three potential phosphorylation residues (S or T) are indicated (bold and underlined). B. Averaged Ca2+-activated currents (at −50 mV) of WT TRPP3 and indicated mutants expressed in three batches of oocytes. ∗∗∗p < 0.001. C. Surface and total expression of WT TRPP3 and mutants in oocytes determined by biotinylation, with β-actin as a loading control, representative of three independent experiments. Alanine and aspartate substitutions mimic dephosphorylation and phosphorylation, respectively. D. Effects of CaMK2 T286D on the function of WT TRPP3 and the T591A mutant assessed by Ca2+-activated currents (at −50 mV) averaged from three batches of oocytes. ∗∗∗p < 0.001; ns: not significant. E. Effects of mutations at site 591 on the TRPP3/CaM binding determined by CaM-Agarose Pull-down. Upper panel: representative data showing the effects of the T591A and T591E mutations. Lower panel: data from three independent experiments were quantified, normalized, and averaged. ns: not significant. F. Effects of CaM on the function of WT TRPP3 and mutant T591A assessed by Ca2+-activated currents (at −50 mV) averaged from three batches of oocytes. ∗∗∗p < 0.001; ns: not significant.

References [+] :

Alberdi, Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation. 2015, Pubmed