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Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators

  • Ion Channels, Receptors and Transporters
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Abstract

The transient receptor potential channel of the ankyrin-binding repeat subfamily, TRPA1, is a Ca2+-permeable non-selective cation channel that depolarizes the plasma membrane and causes Ca2+ influx. A typical feature of TRPA1 is its rapid desensitization following activation by agonists such as mustard oil (MO), cinnamaldehyde, and a high intracellular Ca2+ concentration. In whole-cell recordings on Chinese hamster ovary (CHO) cells expressing TRPA1, desensitization was delayed when phosphatidylinositol 4,5-biphosphate (PIP2) was supplemented via the patch pipette, whereas the PIP2 scavenger neomycin accelerated desensitization. Preincubation with the PI-4 kinase inhibitor wortmannin reduced both constitutive TRPA1 channels activity and the response to MO. Run down was also accelerated by high intracellular Mg2+ concentrations, whereas chelating intracellular Mg2+ with 10 mM ethylenedinitrilotetraacetic acid (EDTA) increased the basal channel activity. In inside-out patches, we observed a rapid run down of TRPA1 activity, which could be prevented by application of diC8-PIP2 or 2 mM Mg-ATP but not Na2-ATP to the cytosolic side of the excised patches. In isolated trigeminal ganglion neurons, preincubation with wortmannin resulted in inhibition of endogenous TRPA1 activation by MO. Taken together, our data indicate that PIP2 modulates TRPA1, albeit to a lesser extent than other known PIP2-dependent TRP channels, and that tools modifying the plasma membrane PIP2 content often have direct effects on this channel.

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Acknowledgments

We thank Drs. A. Segal, R. Vennekens, K. Talavera, D. D'hoedt, N. Damann, and T. Shimizu for very helpful discussions and A. Janssens and M. Benoit for technical assistance. This work was supported by the Human Frontiers Science Programme (HFSP Research Grant Ref. RGP 32/2004), the Belgian Federal Government, the Flemish Government, the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0214.99, F.W.O. G. 0136.00; F.W.O. G.0172.03, Interuniversity Poles of Attraction Program, Prime Ministers Office IUAP) and the Excellentiefinanciering (EF/95/010).

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  1. KU Leuven Department of Mol. Cell Biology, Lab Ion Channel Research, Campus Gasthuisberg, Herestraat 49, bus 802, B-3000, Leuven, Belgium

    Yuji Karashima, Jean Prenen, Grzegorz Owsianik, Thomas Voets & Bernd Nilius

  2. Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez-Consejo Superior de Investigaciones Cientificas, 03550, San Juan de Alicante, Spain

    Victor Meseguer

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  1. Yuji Karashima
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Correspondence to Bernd Nilius.

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Supplementary Figure 1

Effect of PAO on whole-cell currents through TRPA1. (A) Time course of whole-cell currents at +80 mV (open squares) and −80 mV (open circles) elicited by 1 μM PAO. (B) Current–voltage (I–V) relations obtained at the indicated time shown in (A) (GIF 33.5 kb).

High resolution image (TIF 484 KB)

Supplementary Figure 2

Effect of Mg2+ on TRPA1 activity in inside-out patches. In this examples, bath solution contained (in mM) 150 CsCl, 1 MgCl2, 1 sodium triphosphate, 5 EGTA, 10 glucose and 10 HEPES. [Ca2+]i was buffered at 250 nM. Pipette solution contained (in mM) 150 NaCl, 1 MgCl2, 10 glucose, and 10 HEPES. Application of 10 mM Mg2+ immediately inhibited channel activity which slowly recovered. Note that in these experiments we excised the patch in sodium triphosphate PPPi to delay channel run down as described elsewhere [1]. In all five tested cells, 10 mM Mg2+ nearly completely abolished channel activity (before excision P open = 0.42 ± 0.12, during Mg2+ application P open = 0.002 ± 0.001, after wash out of Mg2+ channel activity partially recovered to P open = 0.09±0.01) (GIF 91.7 kb).

High resolution image (TIF 854 KB)

Supplementary Figure 3

Desensitization of TRPA1 also occurs in cell-attached patches. (A) Single channel currents through TRPA1 were measured in cell-attached patches. The averaged current was obtained from three consecutive sweeps at −80 mV. Note the fast current activation by application of MO from outside and the following decay. Channels were constitutive active before application of MO (point a, and panel (B), a). (B) Individual single channel currents during voltage ramps from −100 to +100 mV obtained at the time points marked in (A) (GIF 116 kb).

High resolution image (TIF 669 KB)

Supplementary Figure 4

Activation of TRPA1 by MO in inside-out patches. (A) Single channel currents through TRPA1 were measured after excision in an inside-out patch. Channel activity remained more than 3 min after excision (single channel patch, +60 mV, γ = 90 pS). (B) At +60 mV, open probability was 0.21, mean open time 4.3 ms and closed could only be described by two exponentials indicating long closings between burst-like openings. (C) Application of 200 μM MO to the inner side of the excised patch induced clearly channel activation (+60 mV, γ = 88 pS). (D) Open probability increased to 0.82 mainly due to the disappearance of long closings (same results obtained in six patches). Data indicate that at least in a fraction of excised patches MO can still activate TRPA1 at variance with [1] (GIF 138 kb).

High resolution image (TIF 949 KB)

Supplementary Figure 5

Effect of polylysin on TRPA1 activity in inside-out patches. (A) Single channel openings in a patch clamped at −60 mV and the resulting amplitude histogram (γ = 91 pS, P open increased from 0.43 to 0.92, obtained from the Gaussian fits). (B) Distribution of the open times fitted with a bi-exponential function. A short mean open time of 2.8 ms and a long mean open time of 44.9 ms were calculated. Distribution of the closed times. Note that also two components were found. (C) Same patch and same protocol as in (A) but polylysin is applied to the inner side of the membrane. Note the increased open probability. (D) The open time distribution shows an increased fraction of long openings in the presence of polylysin. The closed time distribution lacks the long closings. This effect causes the increase in open probability in the presence of polylysin via an unknown mechanism (GIF 143 kb).

High resolution image (TIF 960 KB)

Supplementary Figure 6

Predicted topology of TRPA1. (A) Cartoon of the TRPA1 structure. The 14 ANK domains are shown in the N-terminus and pleckstrin homology (PH)-like domains are indicated by the blue bars. PH-like domains are predicted according to the consensus sequence: [R/K]-X3–11-[R/K]-X-[R/K]-[R/K] (2). The TRP box-like motif (L995WYLRK) shares a partial homology to TRP boxes of other TRP channels (see for details on TRP boxes 3). (B) Alignment of the PH-like domains with respective domains found in TRPM4 (2). References: (1) Kim D, Cavanaugh EJ (2007) Requirement of a soluble intracellular factor for activation of transient receptor potential A1 by pungent chemicals: role of inorganic polyphosphates. Journal of NeuroScience 27:6500–9. (2) Nilius B, Mahieu F, Prenen J, Janssens A, Owsianik G, Vennekens R, Voets T (2006) The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate. EMBO Journal 25:467–78. (3) Owsianik G, D’Hoedt D, Voets T, Nilius B (2006) Structure–function relationship of the TRP channel superfamily. Reviews of Physiology Biochemistry and Pharmacology 156:61–90. (GIF 48.4 kb)

High resolution image (TIF 681 KB)

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Karashima, Y., Prenen, J., Meseguer, V. et al. Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators. Pflugers Arch - Eur J Physiol 457, 77–89 (2008). https://doi.org/10.1007/s00424-008-0493-6

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