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A TRPA1-dependent mechanism for the pungent sensation of weak acids.

Wang YY, Chang RB, Allgood SD, Silver WL, Liman ER - J. Gen. Physiol. (2011)

Bottom Line: Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids.Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice.We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Section of Neurobiology, University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

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Responses of TRPA1 to weak acids self-desensitize and cross-desensitize responses to Cin. Whole cell currents evoked in HEK-293 cells expressing TRPA1. (A) TRPA1 currents evoked in response to 10 mM of acetic acid, pH 5, decayed after activation and could not be evoked again by acetic acid, indicating that the channels had entered an inactivated state. (B) After inactivation by 10 mM of acetic acid, pH 5, TRPA1 currents could not be activated by 100 µM Cin, indicating that acetic acid could cross-desensitize responses to Cin. (C) 100 µM Cin elicited large currents in TRPA1-expressing cells that were not preexposed to acetic acid. Currents were recorded in the absence of extracellular Ca2+. Average data are shown below each representative trace. Data are represented by the mean ± SEM. Significance was determined with the two-tailed paired (A; comparison between responses to the first and second application of acetic acid) or unpaired (B and C; comparison between responses to Cin with or without preexposure to acetic acid) Student’s t test. *, P < 0.05; ***, P < 0.001.
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fig6: Responses of TRPA1 to weak acids self-desensitize and cross-desensitize responses to Cin. Whole cell currents evoked in HEK-293 cells expressing TRPA1. (A) TRPA1 currents evoked in response to 10 mM of acetic acid, pH 5, decayed after activation and could not be evoked again by acetic acid, indicating that the channels had entered an inactivated state. (B) After inactivation by 10 mM of acetic acid, pH 5, TRPA1 currents could not be activated by 100 µM Cin, indicating that acetic acid could cross-desensitize responses to Cin. (C) 100 µM Cin elicited large currents in TRPA1-expressing cells that were not preexposed to acetic acid. Currents were recorded in the absence of extracellular Ca2+. Average data are shown below each representative trace. Data are represented by the mean ± SEM. Significance was determined with the two-tailed paired (A; comparison between responses to the first and second application of acetic acid) or unpaired (B and C; comparison between responses to Cin with or without preexposure to acetic acid) Student’s t test. *, P < 0.05; ***, P < 0.001.

Mentions: For Ca2+-imaging experiments, the bath solution was: 150 mM NaCl, 10 mM HEPES, and 2 mM CaCl2, pH 7.4. HEPES-buffered acidic solution was bath solution adjusted to pH 6.5. PA solution contained: 100 mM PA, 60 mM NaCl, and 2 mM CaCl2, pH 6.5. High K+ solution was 150 mM KCl, 10 mM HEPES, and 2 mM CaCl2, pH 7.4. For intracellular pH imaging and whole cell and cell-attached recordings, the bath solution was (unless otherwise stated): 150 mM NaCl, 0.5 mM EGTA, and 10 mM HEPES, pH 7.4. Acidic solutions contained: 150 mM NaCl, 0.5 mM EGTA, and either 10 mM HEPES, pH 5, 10 mM MES, pH 5, or 10 mM acetic acid, pH 5, 6, or 7. Other solutions containing weak acids were similarly composed, and where the concentration of the acid was >10 mM, the concentration of NaCl was decreased accordingly. Internal solution contained: 145 mM CsCl, 5 mM EGTA, 3 mM CaCl2 (100 nM free Ca2+), 2 mM MgATP, and 10 mM HEPES, pH 7.4 with CsOH. To exclude the possibility that the observed effects were a result of release of Ca2+ from the chelator in response to intracellular acidification, in some experiments, Ca2+ and MgATP were excluded from the internal solution (as stated and Fig. 6). For cell-attached and excised patch experiments, the pipette solution contained: 150 mM NaCl, 10 mM HEPES, and 0.5 mM EGTA, pH 7.4. For excised patch experiments, solution applied to the cytoplasmic side of the cell contained: 150 mM KCl, 0.5 mM EGTA, and 1 mM pentasodium tripolyphosphate hexahydrate (polyP3) (Kim and Cavanaugh, 2007) buffered with either 10 mM HEPES, pH 7.4, or 10 mM MES, pH 5.5, or 10 mM acetic acid, pH 7.3.


A TRPA1-dependent mechanism for the pungent sensation of weak acids.

Wang YY, Chang RB, Allgood SD, Silver WL, Liman ER - J. Gen. Physiol. (2011)

Responses of TRPA1 to weak acids self-desensitize and cross-desensitize responses to Cin. Whole cell currents evoked in HEK-293 cells expressing TRPA1. (A) TRPA1 currents evoked in response to 10 mM of acetic acid, pH 5, decayed after activation and could not be evoked again by acetic acid, indicating that the channels had entered an inactivated state. (B) After inactivation by 10 mM of acetic acid, pH 5, TRPA1 currents could not be activated by 100 µM Cin, indicating that acetic acid could cross-desensitize responses to Cin. (C) 100 µM Cin elicited large currents in TRPA1-expressing cells that were not preexposed to acetic acid. Currents were recorded in the absence of extracellular Ca2+. Average data are shown below each representative trace. Data are represented by the mean ± SEM. Significance was determined with the two-tailed paired (A; comparison between responses to the first and second application of acetic acid) or unpaired (B and C; comparison between responses to Cin with or without preexposure to acetic acid) Student’s t test. *, P < 0.05; ***, P < 0.001.
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Related In: Results  -  Collection

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fig6: Responses of TRPA1 to weak acids self-desensitize and cross-desensitize responses to Cin. Whole cell currents evoked in HEK-293 cells expressing TRPA1. (A) TRPA1 currents evoked in response to 10 mM of acetic acid, pH 5, decayed after activation and could not be evoked again by acetic acid, indicating that the channels had entered an inactivated state. (B) After inactivation by 10 mM of acetic acid, pH 5, TRPA1 currents could not be activated by 100 µM Cin, indicating that acetic acid could cross-desensitize responses to Cin. (C) 100 µM Cin elicited large currents in TRPA1-expressing cells that were not preexposed to acetic acid. Currents were recorded in the absence of extracellular Ca2+. Average data are shown below each representative trace. Data are represented by the mean ± SEM. Significance was determined with the two-tailed paired (A; comparison between responses to the first and second application of acetic acid) or unpaired (B and C; comparison between responses to Cin with or without preexposure to acetic acid) Student’s t test. *, P < 0.05; ***, P < 0.001.
Mentions: For Ca2+-imaging experiments, the bath solution was: 150 mM NaCl, 10 mM HEPES, and 2 mM CaCl2, pH 7.4. HEPES-buffered acidic solution was bath solution adjusted to pH 6.5. PA solution contained: 100 mM PA, 60 mM NaCl, and 2 mM CaCl2, pH 6.5. High K+ solution was 150 mM KCl, 10 mM HEPES, and 2 mM CaCl2, pH 7.4. For intracellular pH imaging and whole cell and cell-attached recordings, the bath solution was (unless otherwise stated): 150 mM NaCl, 0.5 mM EGTA, and 10 mM HEPES, pH 7.4. Acidic solutions contained: 150 mM NaCl, 0.5 mM EGTA, and either 10 mM HEPES, pH 5, 10 mM MES, pH 5, or 10 mM acetic acid, pH 5, 6, or 7. Other solutions containing weak acids were similarly composed, and where the concentration of the acid was >10 mM, the concentration of NaCl was decreased accordingly. Internal solution contained: 145 mM CsCl, 5 mM EGTA, 3 mM CaCl2 (100 nM free Ca2+), 2 mM MgATP, and 10 mM HEPES, pH 7.4 with CsOH. To exclude the possibility that the observed effects were a result of release of Ca2+ from the chelator in response to intracellular acidification, in some experiments, Ca2+ and MgATP were excluded from the internal solution (as stated and Fig. 6). For cell-attached and excised patch experiments, the pipette solution contained: 150 mM NaCl, 10 mM HEPES, and 0.5 mM EGTA, pH 7.4. For excised patch experiments, solution applied to the cytoplasmic side of the cell contained: 150 mM KCl, 0.5 mM EGTA, and 1 mM pentasodium tripolyphosphate hexahydrate (polyP3) (Kim and Cavanaugh, 2007) buffered with either 10 mM HEPES, pH 7.4, or 10 mM MES, pH 5.5, or 10 mM acetic acid, pH 7.3.

Bottom Line: Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids.Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice.We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Section of Neurobiology, University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

Show MeSH
Related in: MedlinePlus