Pore dilation occurs in TRPA1 but not in TRPM8 channels
© Chen et al; licensee BioMed Central Ltd. 2009
Received: 20 October 2008
Accepted: 21 January 2009
Published: 21 January 2009
Abundantly expressed in pain-sensing neurons, TRPV1, TRPA1 and TRPM8 are major cellular sensors of thermal, chemical and mechanical stimuli. The function of these ion channels has been attributed to their selective permeation of small cations (e.g., Ca2+, Na+ and K+), and the ion selectivity has been assumed to be an invariant fingerprint to a given channel. However, for TRPV1, the notion of invariant ion selectivity has been revised recently. When activated, TRPV1 undergoes time and agonist-dependent pore dilation, allowing permeation of large organic cations such as Yo-Pro and NMDG+. The pore dilation is of physiological importance, and has been exploited to specifically silence TRPV1-positive sensory neurons. It is unknown whether TRPA1 and TRPM8 undergo pore dilation. Here we show that TRPA1 activation by reactive or non-reactive agonists induces Yo-Pro uptake, which can be blocked by TRPA1 antagonists. In outside-out patch recordings using NMDG+ as the sole external cation and Na+ as the internal cation, TRPA1 activation results in dynamic changes in permeability to NMDG+. In contrast, TRPM8 activation does not produce either Yo-Pro uptake or significant change in ion selectivity. Hence, pore dilation occurs in TRPA1, but not in TRPM8 channels.
Abundantly expressed in sensory neurons, TRPV1, TRPA1 and TRPM8 are involved in sensory function, pain and neurogenic inflammation . The function of these ion channels has been attributed to their ability to pass certain ion species across the plasma membrane. Once activated, TRPV1, TRPA1 and TRPM8 are permeable to small cations such as Ca2+, K+, Na+; hence, channel activation simultaneously depolarizes the plasma membrane and raises intracellular Ca2+, which subsequently triggers a variety of physiological processes. By analogy to voltage-gated K+ channels, it is assumed that ion selectivity of TRP channels should be an invariant signature to the respective channel. However, this notion has been challenged recently. When activated, TRPV1 exhibits time and agonist-dependent changes in ion selectivity . In fact, TRPV1 undergoes pore dilation and allows permeation of large organic cations, including spermine (202.3 Da), NMDG (195.2 Da), Yo-Pro (376 Da), gentamycin (477.6 Da) and QX-314 [3–7]. Here we explored whether TRPA1 and TRPM8 undergo pore dilation by examining Yo-Pro uptake and changes in ion selectivity upon channel activation.
Results and discussion
Several small molecule inhibitors of TRPA1 have been described recently, including AP18, HC-030031 and ruthenium red (RR) [15, 16]. We tested whether these antagonists blocked Yo-Pro uptake. AP18 attenuated 30 μM AITC-induced Yo-Pro uptake in a concentration-dependent manner, with an IC50 of 10.3 ± 0.8 μM (Fig. 2C and 2D). Likewise, HC-030031 and RR also completely blocked Yo-Pro uptake (IC50: 9.4 ± 0.6 μM for HC-030031 and 10.0 ± 1.6 μM for RR). Taken together, these data show that agonist-evoked Yo-Pro uptake is related to TRPA1 channel activities.
The accuracy of Erev measurement could be compromised by small current amplitudes, especially for basal currents and currents immediately following AITC application. However, the Erev of basal currents was consistent across patches (-95.3 ± 4.8 mV, n = 5), and Erev shifts consistently occurred in TRPA1, but not in TRPM8. In addition, even for relatively large TRPA1 currents, significant shifts in Erev occurred. For example, the shift in Erev was 31.6 mV between 6 s and 15 s pulses, and 14.2 mV between 9 s and 15 s pulses (Fig. 3A and 4A). Taken together, these data suggests that the dynamic change in Erev results from TRPA1 channel activity. Another concern in extrapolating the change in Erev to the change in ion selectivity is that ion accumulation can occur during prolonged activation, particularly when large currents are conducted under whole cell configuration. However, the ion accumulation should not significantly compromise our TRPA1 experiments using outside-out patch configuration, in which extracellular and intracellular ionic conditions were well controlled. In addition, reversal potentials changed within seconds of AITC application when currents were small, but reached a steady state (from 15 to 60 s) when currents were relatively large (Fig. 3A and 4A). Furthermore, TRPM8 conducted currents with similar amplitudes but without significant shifts in reversal potential. Consistent with the electrophysiology data, the large divalent cation Yo-Pro did not cross the membrane when the channel was closed or blocked by antagonists, but permeated the membrane freely when the channel was open (Fig. 1B and 2C). Collectively, our data suggest that TRPA1, but not TRPM8, undergoes pore dilation.
Pore dilation has been previously described for the ATP-gated P2X and TRPV1 [2, 8, 9]. For P2X channels, the mechanism underlying pore dilation remains controversial. Several alternative mechanisms have been proposed including a direct change in ion selectivity, formation of channel multimers, and recruitment of a downstream, nonselective pore [19, 20]. For TRPV1, pore dilation most likely arises from a change in ion selectivity, as indicated by the dynamic change in ion selectivity during agonist stimulation, and the effects of mutations and chemical modification of certain residues within the selectivity filter . In the current study, we did not elucidate the biophysical mechanism underlying pore dilation of TRPA1. However, there were several notable observations. First, the permeability to NMDG+ increased almost immediately upon channel activation. Second, the outside-out patch configuration should largely disrupt cytoskeletal structures and washout cytosolic factors. Third, the AITC evoked- NMDG+ and Na+ conductance was sensitive to blockade by ruthenium red. Finally, under identical conditions, TRPM8 conducted large currents, but did not exhibit Yo-Pro uptake or a significant change in NMDG+ permeability. Thus, TRPA1 pore dilation most likely represents a direct change in ion selectivity. Nonetheless, our present study does not completely rule out the involvement of other proteins.
TRPV1, TRPA1 and TRPM8 are major TRP channels involved in somatosensation. Within dorsal root ganglia, TRPV1 and TRPA1 are co-expressed and interact functionally in one population of sensory neurons, while TRPM8 is expressed largely in a separate neuronal population. Interestingly, pore dilation occurs in TRPA1, TRPV1 but not TRPM8, suggesting that this property is not ubiquitous, but rather specific to subtypes of channels within a subpopulation of neurons. The change in cation permeability, in turn, may alter channel function, affect a host of downstream processes (e.g., neurotransmitter release, cellular toxicity) and contribute to pain hypersensitivity . Recently, it was reported that TRPV1-mediated pore dilation could be utilized to deliver QX-314 (a membrane-impermeant sodium channel blocker) specifically to TRPV1-positive sensory neurons, achieving analgesic effects without motor deficits associated with local anesthetics . However, this strategy of targeting TRPV1-positive neurons could be compromised by several factors, including the broad expression pattern of TRPV1, its role in regulating body temperature, and its involvement in hippocampal synaptic plasticity [22, 23]. By analogy to TRPV1, the pore dilation of TRPA1 could be exploited to mediate entry of QX-314 specifically into TRPA1-positive neurons. Given the restrictive expression of TRPA1 in sensory neurons, this strategy may offer analgesic efficacy without unwanted side effects.
In conclusion, the present study demonstrates that pore dilution occurs in TRPA1 but not in TRPM8 channels. This finding raises many interesting questions: What is the exact biophysical mechanism underlying pore dilation of TRPA1? What are the physiological, pathological and therapeutic implications? Why does pore dilation not occur in TRPM8? What are the pore behaviors of other TRP channels? Answers to these questions will certainly extend our understanding of this family of ion channels.
Transient expression of recombinant TRPs
Full length cDNAs for rat TRPA1(GenBank Accession: NM_207608), human TRPA1 (NM_007332), mouse TRPA1 (NM_177781) and human TRPM8 (NM_024080) were cloned into pcDNA3.1/V5-His TOPO vector and transiently expressed in HEK293-F or HeLa cells . For the Ca2+ influx or Yo-Pro uptake assay, HEK293-F cells were transfected with TRP cDNA, collected 48 hours post transfection, and used either fresh or following storage at -70°C. For electrophysiological experiments, HeLa cells were transfected with TRPA1 or TRPM8 plus GFP, and used 48 hours later.
Ca2+ influx and Yo-Pro uptake assays
Ca2+ influx assay was performed using the FLIPR™ and calcium assay kit R8033 (MDS Analytical Technology) as reported previously . After incubation with 100 μl of 1 × Ca2+ dye for ~2 hours at room temperature, a two-addition protocol was used for evaluating agonist activities (i.e., activation of Ca2+ influx) and antagonist activities (i.e., inhibition of agonist responses): 10 s baseline readout, addition of 50 μl assay buffer or antagonist (4 × stock), 3–4 min readout, addition of 50 μl agonist (4 × stock), and readout for 2.5 min. Maximum minus minimum signals before the second addition and at the end of the experiment were obtained.
Yo-Pro uptake was determined using the FLIPR™ and Mg2+/Ca2+-free DPBS buffer as reported previously . Briefly, immediately after loading with 100 μl Yo-Pro dye (2 μM), a two-addition protocol was used for evaluating agonist activity (i.e., Yo-Pro uptake) and antagonist activities (i.e., inhibition of agonist evoked Yo-Pro uptake): 10 s baseline readout, addition of 50 μl assay buffer or antagonist, 3 min readout, addition of 50 μl agonists, and readout for 60 min. Max-min fluorescence signals before the second addition and at the end of the experiment were obtained.
Outside-out patch recording
Before forming cell-attached patches from HeLa cells expressing TRPA1 or TRPM8, pipette offset was adjusted to give a zero current value. Outside-out patches with the least amount of leak were used, as judged by the very small DC shift (<5 mV) of the basal current at different membrane potentials. Currents were recorded using AxoPatch200B. The pipette solution contained (mM): 140 NaCl, 1 MgCl2, 5 mM EGTA, and 10 HEPES (pH 7.3). For experiments with NMDG+, the bath solution contained (mM): 150 mM NMDG+, 115 mM Cl-, 5 mM EGTA, and 10 HEPES (pH 7.3). For Cl- replacement experiments, the bath contained 150 mM NMDG+, 62 mM EGTA and 10 HEPES (pH 7.3). Patch membrane potential was held at -80 mV, and then a voltage ramp from -140 mV to 0 mV (500 ms duration) was applied every 3 seconds. Current was filtered at 1 kHz using 8-pole Bessel filter (-3 dB; Frequency Devices) and transferred directly to a computer using the Digidata 1320 interface (Axon Instruments) at a sampling rate of 10 kHz. Permeability ratio (PX/PNa) was calculated using the equation: PX/PNa = ([X]o/[Na]o)• exp(ΔErev •F/RT); where ΔErev represents the shift in Erev after addition of AITC in NMDG+ external/Na+ internal solution, and F/RT is 0.040 mV-1. The activity coefficient of Na+ and NMDG+ was taken as 0.75 and 0.81, respectively. Student's t-test was used with p < 0.05 as the criterion for significance. Data are represented as mean ± S.E. unless specified otherwise.
- Wang H, Woolf CJ: Pain TRPs. Neuron 2005,46(1):9–12. 10.1016/j.neuron.2005.03.011PubMedView Article
- Chung MK, Guler AD, Caterina MJ: TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nat Neurosci 2008,11(5):555–64. 10.1038/nn.2102PubMedView Article
- Ahern GP, Wang X, Miyares RL: Polyamines are potent ligands for the capsaicin receptor TRPV1. J Biol Chem 2006,281(13):8991–5. 10.1074/jbc.M513429200PubMedView Article
- Meyers JR, MacDonald RB, Duggan A, Lenzi D, Standaert DG, Corwin JT, Corey DP: Lighting up the senses: FM1–43 loading of sensory cells through nonselective ion channels. J Neurosci 2003,23(10):4054–65.PubMed
- Binshtok AM, Bean BP, Woolf CJ: Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature 2007,449(7162):607–10. 10.1038/nature06191PubMedView Article
- Myrdal SE, Steyger PS: TRPV1 regulators mediate gentamicin penetration of cultured kidney cells. Hear Res 2005,204(1–2):170–82. 10.1016/j.heares.2005.02.005PubMed CentralPubMedView Article
- Hellwig N, Plant TD, Janson W, Schafer M, Schultz G, Schaefer M: TRPV1 acts as proton channel to induce acidification in nociceptive neurons. J Biol Chem 2004,279(33):34553–61. 10.1074/jbc.M402966200PubMedView Article
- Virginio C, MacKenzie A, Rassendren FA, North RA, Surprenant A: Pore dilation of neuronal P2X receptor channels. Nat Neurosci 1999,2(4):315–21. 10.1038/7225PubMedView Article
- Khakh BS, Bao XR, Labarca C, Lester HA: Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nat Neurosci 1999,2(4):322–30. 10.1038/7233PubMedView Article
- Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A: ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003,112(6):819–29. 10.1016/S0092-8674(03)00158-2PubMedView Article
- Niforatos W, Zhang XF, Lake MR, Walter KA, Neelands T, Holzman TF, Scott VE, Faltynek CR, Moreland RB, Chen J: Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597). Mol Pharmacol 2007,71(5):1209–16. 10.1124/mol.106.033621PubMedView Article
- Cavanaugh EJ, Simkin D, Kim D: Activation of transient receptor potential A1 channels by mustard oil, tetrahydrocannabinol and Ca(2+) reveals different functional channel states. Neuroscience 2008,154(4):1467–76. 10.1016/j.neuroscience.2008.04.048PubMedView Article
- Maher M, Ao H, Banke T, Nasser N, Wu NT, Breitenbucher JG, Chaplan SR, Wickenden AD: Activation of TRPA1 by farnesyl thiosalicylic acid. Mol Pharmacol 2008,73(4):1225–34. 10.1124/mol.107.042663PubMedView Article
- Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, Imamachi N, Andre E, Patacchini R, Cottrell GS, Gatti R, Basbaum AI, Bunnett NW, Julius D, Geppetti P: 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci USA 2007,104(33):13519–24. 10.1073/pnas.0705923104PubMed CentralPubMedView Article
- Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, Jegla T, Patapoutian A: A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol Pain 2007, 3: 40. 10.1186/1744-8069-3-40PubMed CentralPubMedView Article
- McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM: TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci USA 2007,104(33):13525–30. 10.1073/pnas.0705924104PubMed CentralPubMedView Article
- Nagata K, Duggan A, Kumar G, Garcia-Anoveros J: Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J Neurosci 2005,25(16):4052–61. 10.1523/JNEUROSCI.0013-05.2005PubMedView Article
- Zhang XF, Chen J, Faltynek CR, Moreland RB, Neelands TR: Transient receptor potential A1 mediates an osmotically activated ion channel. Eur J Neurosci 2008,27(3):605–11. 10.1111/j.1460-9568.2008.06030.xPubMedView Article
- Fujiwara Y, Kubo Y: Density-dependent changes of the pore properties of the P2X2 receptor channel. J Physiol 2004,558(Pt 1):31–43. 10.1113/jphysiol.2004.064568PubMed CentralPubMedView Article
- Locovei S, Scemes E, Qiu F, Spray DC, Dahl G: Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex. FEBS Lett 2007,581(3):483–8. 10.1016/j.febslet.2006.12.056PubMed CentralPubMedView Article
- Bautista D, Julius D: Fire in the hole: pore dilation of the capsaicin receptor TRPV1. Nat Neurosci 2008,11(5):528–9. 10.1038/nn0508-528PubMedView Article
- Gavva NR, Bannon AW, Surapaneni S, Hovland DN Jr, Lehto SG, Gore A, Juan T, Deng H, Han B, Klionsky L, Kuang R, Le A, Tamir R, Wang J, Youngblood B, Zhu D, Norman MH, Magal E, Treanor JJ, Louis JC: The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation. J Neurosci 2007,27(13):3366–74. 10.1523/JNEUROSCI.4833-06.2007PubMedView Article
- Gibson HE, Edwards JG, Page RS, Van Hook MJ, Kauer JA: TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons. Neuron 2008,57(5):746–59. 10.1016/j.neuron.2007.12.027PubMed CentralPubMedView Article
- Chen J, Zhang XF, Kort ME, Huth JR, Sun C, Miesbauer LJ, Cassar SC, Neelands T, Scott VE, Moreland RB, Reilly RM, Hajduk PJ, Kym PR, Hutchins CW, Faltynek CR: Molecular determinants of species-specific activation or blockade of TRPA1 channels. J Neurosci 2008,28(19):5063–71. 10.1523/JNEUROSCI.0047-08.2008PubMedView Article
- Chen J, Lake MR, Sabet RS, Niforatos W, Pratt SD, Cassar SC, Xu J, Gopalakrishnan S, Pereda-Lopez A, Gopalakrishnan M, Holzman TF, Moreland RB, Walter KA, Faltynek CR, Warrior U, Scott VE: Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists. J Biomol Screen 2007,12(1):61–9. 10.1177/1087057106295220PubMedView Article
- Donnelly-Roberts DL, Namovic MT, Faltynek CR, Jarvis MF: Mitogen-activated protein kinase and caspase signaling pathways are required for P2X7 receptor (P2X7R)-induced pore formation in human THP-1 cells. J Pharmacol Exp Ther 2004,308(3):1053–61. 10.1124/jpet.103.059600PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.