Canna~Fangled Abstracts

Activation of transient receptor potential A1 channels by mustard oil, tetrahydrocannabinol and Ca2+ reveals different functional channel states.

By October 31, 2013No Comments

pm2Activation of transient receptor potential A1 channels by mustard oil, tetrahydrocannabinol and Ca2+ reveals different functional channel states.

Source

Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064, USA.

Abstract

Our recent study has shown that activation of transient receptor potential A1 channel (TRPA1) by pungent chemicals such as allyl-isothiocyanate (AITC) requires an unidentified cytosolic factor whose action can be mimicked by inorganic polyphosphates. Thus, AITC and other pungent chemicals fail to activate TRPA1 in excised patches. It is unclear whether TRPA1 switches to a conformation that is insensitive to the pungent chemicals, or whether TRPA1 simply becomes completely non-functional and insensitive to all activators when the cytosolic factor is absent. To help distinguish between these possibilities, the effects of Delta(9)-tetrahydrocannabinol (THC) and Ca(2+) that are structurally different from pungent chemicals were tested on AITC-sensitive and AITC-insensitive states of TRPA1. In HeLa cells transiently expressing mouse TRPA1, activation of TRPA1 by THC was slow and weak from the extracellular side (cell-attached; K(1/2) >20 microM), but was faster and more potent from the intracellular side (inside-out; K(1/2), approximately 0.7 microM), and this did not require the presence of a polyphosphate. Similar results were observed in rat trigeminal neurons. Increasing the extracellular [Ca(2+)] from approximately 0-1-3 mM activated TRPA1 in cell-attached patches. Elevation of cytosolic [Ca(2+)] using thapsigargin (inhibitor of Ca(2+)-ATPAse) and histamine (that elevates IP(3)) also activated TRPA1 in cell-attached patches. Similar to pungent chemicals, Ca(2+) (1-5 microM) failed to activate TRPA1 in inside-out patches, unless polyphosphates were present. These results show that TRPA1 can exist in different functional states: a native state (cell-attached patch) and a non-native state (excised patch). THC can activate TRPA1 even in the absence of polyphosphates, whereas pungent chemicals and Ca(2+) require it for activation.
PMID:

 18515013
[PubMed – indexed for MEDLINE]

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Abbreviations

  • AITC, allyl-isothiocyanate;
  • DMEM, Dulbecco’s modified Eagle’s medium;
  • ER, endoplasmic reticulum;
  • GFP, green fluorescent protein;
  • TG, trigeminal ganglion;
  • THC, Δ9-tetrahydrocannabinol;
  • TRPA1, transient receptor potential A1 channel

Figures and tables from this article:

Full-size image (39 K)
Fig. 1.

Activation of TRPA1 by AITC and THC. Cell-attached and inside-out configurations are shown for each tracing. Unless indicated otherwise, the pipette potential was held at +40 mV to record inward current. Mean and standard deviation ofNPo values are shown in parentheses. (A) Cell-attached patch was formed on HeLa cells expressing TRPA1. AITC applied to the bath solution activated the channels (NPo; 6.2±1.8; n=5). Formation of inside-out patch closed all channels. (B) THC applied to the bath solution of cell-attached patches did not activate TRPA1. Additional application of AITC activated the channels (NPo, 6.8±1.6; n=5). (C) Cell-attached patch was formed and THC applied to the bath solution. Formation of inside-out patch activated TRPA1 at pipette potentials of −60 (outward current; NPo, 5.3±1.6; n=4) and +40 mV (inward current; NPo, 4.2±1.4; n=4). (D) THC was applied to the cytoplasmic side of the inside-out patch (NPo, 5.1±1.4; n=4). (E) AITC applied to the inside-out patch did not activate TRPA1. Further addition of THC activated TRPA1 (NPo, 6.1±2.1; n=4).

Full-size image (41 K)
Fig. 2.

Properties of single channels activated by THC. (A) THC was applied to inside-out patch to activate TRPA1, and single channel openings recorded at various membrane potentials. (B) Amplitude histogram of openings recorded at −40 mV is shown. (C) The plot shows the current–voltage relationship of the largest conductance. Each point is the mean±S.D. of three determinations. (D) The presence of THC in the pipette of outside-out patch caused activation of TRPA1 (NPo, 4.3±1.3; n=3). Ruthenium Red was then applied to the bath solution (NPo, 0.11±0.04; n=3). (E) Data from outside-out patches with THC in the pipette are used. Current–voltage relationship obtained before and after application of Ruthenium Red.

Full-size image (49 K)
Fig. 3.

THC does not require polyphosphates to activate TRPA1. (A) Inside-out patch was formed in the presence of 5 mM PPPi in the bath solution. Further addition of AITC caused activation of TRPA1 (NPo, 6.4±1.5; n=5). (B) Inside-out patch was formed in the presence of 5 mM PPPi in the bath solution. Further addition of THC caused activation of TRPA1 (NPo, 7.4±2.2; n=5). Washout of PPPi did not cause a further change in channel activity (NPo, 6.2±1.9; n=5). (C) Inside-out patch was formed and THC added to the bath solution to activate TRPA1 (NPo, 5.4±1.6; n=5). Addition of PPPi produced no further change in channel activity (NPo, 5.6±1.5; n=5). (D, E) Inside-out patches are formed from cells expressing TRPM8 or TRPV1. Application of THC did not activate the channels, but menthol or capsaicin activated its respective targets as shown.

Full-size image (27 K)
Fig. 4.

Concentration-dependent activation of TRPA1 by THC and AITC. (A) Inside-out patch was formed in the presence of 2 mM PPPi in the bath solution. THC or AITC was added in increasing concentrations until a maximum level was achieved. (B) Channel activity was determined for each concentration of the agonist, and then plotted as a function of agonist concentration. The data points were fitted with the Hill equation as described in the text. Each point is the mean±S.D. of four determinations. K1/2 values are 0.67 μM and 15.5 μM for THC and AITC, respectively.

Full-size image (58 K)
Fig. 5.

Activation of TRPA1 by elevation of extracellular Ca2+. (A) Cell-attached patch was formed in Ca2+-free solution. [Ca2+] in the bath solution was then increased to 1 mM for ∼1 min and then washed off. AITC was then added to maximally activate TRPA1. Inset shows single channel openings activated by 1 mM external Ca2+. (B) Same experiments as in A except that 2 mM external Ca2+ was used to activate TRPA1. (C) Same experiments as in A except that 3 mM external Ca2+ was used to activate TRPA1. Here, the pipette potential was switched from +40 mV to −60 mV to record inward and outward currents. (D) Cell-attached patch was formed in solution containing 1 mM external Ca2+. Histamine was then applied to the bath solution to activate the Gq signaling pathway. (E) Cell-attached patch was formed in solution containing 1 mM Ca2+. Thapsigargin was applied to cause a rise in cytosolic [Ca2+]. (F) A graph shows the increase in TRPA1 activity in response to elevation of external [Ca2+] relative to the maximal activation produced by AITC. Each bar is the mean±S.D. of four determinations.

Full-size image (20 K)
Fig. 6.

Direct activation of TRPA1 by an increase in cytosolic [Ca2+]. (A) Inside-out patch was formed in Ca2+-free (<10 nM) solution. [Ca2+] in the bath solution was then increased to 1 μM for ∼1 min and then washed off. AITC was added later. (B) Same experiment as in A except that 5 μM Ca2+ was applied to the bath solution. At the end of each experiment, THC was applied to confirm the presence of TRPA1. (C) In inside-out patch containing 2 mM PPPi in the bath solution, [Ca2+] was increased from ∼0–5 μM. After washout of only Ca2+, AITC was added to cause activation of TRPA1. In the presence of AITC, PPPi was then washed off, resulting in closing of channels. Inset shows the increase in TRPA1 activity in response to 5 μM Ca2+ in the presence of PPPi (n=5). Each bar is the mean±S.D. of four determinations, and asterisk indicates a significant difference (P<0.05).

Full-size image (42 K)
Fig. 7.

Activation of TRPA1 expressed in trigeminal neurons by THC. (A) Cell-attached patch was formed and THC (20 μM) applied to the bath solution first, and then AITC (50 μM) added later to activate TRPA1 (NPo, 2.6±0.6; n=3). Pipette potential was +40 mV. A photomicrograph of TG neurons in culture is shown. Single channel opening at higher time resolution is also shown. (B) Cell-attached patch was formed with THC (20 μM) in the bath solution. After ∼3 min, inside-out patch was formed. This resulted in activation of TRPA1-like channels (NPo, 2.9±0.5; n=4). Pipette potential was +40 mV. (C) Outside-out patch was formed with THC in the pipette, activating TRPA1 (NPo, 1.8±0.3; n=4). Ruthenium Red was then applied to the bath solution (NPo, 0.0±0.0). (D) Inside-out patch was formed with PPPi in the bath solution. Application of 5 μM Ca2+ activated TRPA1 reversibly (NPo, 1.4±0.3; n=4). Subsequent application of AITC caused much greater activation (NPo, 8.4±1.8)

Full-size image (69 K)
Fig. 8.

A scheme showing activation of TRPA1 by AITC and THC in cell-attached and inside-out patches. The cytoplasmic N terminus contains cysteine residues where AITC interacts, but this requires endogenous factor (or polyphosphates) to keep this region in the responsive (native) conformation. The EF hand Ca2+ binding site is found within this region and also requires the native conformation. In the inside-out patch where the cytoplasm and the putative factor are washed out, the AITC region may be collapsed (i.e. altered conformation), preventing AITC from gaining proper access to the cysteine residues for covalent modification. However, the THC site presumed to be also at the N-terminus is unaffected by the washout of the cytosolic factor. Therefore, THC is able to gain access to its binding site without difficulty and activate the channel.

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Corresponding author. Tel: +1-847-578-3280; fax: +1-847-578-3265.

Copyright © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
 
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