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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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TRPA1 is activated by weak acids that acidify the cell cytosol. (A) Changes in emission from the pH-sensitive fluorescent probe carboxy-DFFDA in HEK-293 cells in response to acetic acid at varying concentrations (0, 0.5, 2, 10, and 100 mM at pH 5) and pH (10 mM; pH 5, 6, and 7) and to a panel of carboxylic acids (10 or 100 mM; pH 5). A representative experiment is shown on the left. (B) Proposed model for how acetic acid might activate TRPA1. (C) Currents evoked in HEK-293 cells expressing TRPA1 (peak magnitude at + 80 mV) in response to acetic acid at varying concentrations and pH, and to other carboxylic acids as indicated. Same color scheme as in A. Data for 0 and 10 mM of acetic acid, pH 5, were reproduced from Fig. 2 B. Representative traces of current activation by 100 mM of lactic acid (LA), pH 5, and 10 mM PA, pH 5, are shown on the left. Inset shows the structures of the acids. (D) The magnitude of the TRPA1 current plotted as a function of the change in fluorescence of the pH-sensitive dye carboxy-DFFDA. Colors correspond to the scheme in A and B. The correlation between the change in fluorescence and the magnitude of the TRPA1 current suggests that intracellular pH is the proximate stimulus that gates TRPA1 in response to extracellularly applied weak acids. Data are represented by the mean ± SEM.
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fig3: TRPA1 is activated by weak acids that acidify the cell cytosol. (A) Changes in emission from the pH-sensitive fluorescent probe carboxy-DFFDA in HEK-293 cells in response to acetic acid at varying concentrations (0, 0.5, 2, 10, and 100 mM at pH 5) and pH (10 mM; pH 5, 6, and 7) and to a panel of carboxylic acids (10 or 100 mM; pH 5). A representative experiment is shown on the left. (B) Proposed model for how acetic acid might activate TRPA1. (C) Currents evoked in HEK-293 cells expressing TRPA1 (peak magnitude at + 80 mV) in response to acetic acid at varying concentrations and pH, and to other carboxylic acids as indicated. Same color scheme as in A. Data for 0 and 10 mM of acetic acid, pH 5, were reproduced from Fig. 2 B. Representative traces of current activation by 100 mM of lactic acid (LA), pH 5, and 10 mM PA, pH 5, are shown on the left. Inset shows the structures of the acids. (D) The magnitude of the TRPA1 current plotted as a function of the change in fluorescence of the pH-sensitive dye carboxy-DFFDA. Colors correspond to the scheme in A and B. The correlation between the change in fluorescence and the magnitude of the TRPA1 current suggests that intracellular pH is the proximate stimulus that gates TRPA1 in response to extracellularly applied weak acids. Data are represented by the mean ± SEM.

Mentions: We previously reported that CO2 activates TRPA1 by acidifying the cell cytosol (Wang et al., 2010). Acetic acid and other carboxylic acids are well known to produce intracellular acidification and thus might activate TRPA1 through the same mechanism. Acetic acid has a pKa of 4.88, and at pH 5.0, a substantial portion of the acid (∼37%) is in the protonated membrane-permeable form. In contrast, the standard pH buffers HEPES and MES are both zwitterionic, not membrane permeable at pH 5.0, and not expected to produce intracellular acidification. We confirmed that under the conditions of our experiments, acetic acid produces intracellular acidification by measuring the response of HEK cells loaded with a pH-sensitive fluorescent probe, carboxy-DFFDA. Acidic extracellular solution containing acetic acid and not HEPES or MES produced robust intracellular acidification. Moreover, as expected, increasing the concentration of acetic acid, while holding the extracellular pH constant, produced a more robust and rapid acidification of the cell cytosol. Also, as expected, at more alkaline pH (pH 6 or pH 7), the same concentration of acetic acid produced weaker intracellular acidification (Fig. 3 A).


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)

TRPA1 is activated by weak acids that acidify the cell cytosol. (A) Changes in emission from the pH-sensitive fluorescent probe carboxy-DFFDA in HEK-293 cells in response to acetic acid at varying concentrations (0, 0.5, 2, 10, and 100 mM at pH 5) and pH (10 mM; pH 5, 6, and 7) and to a panel of carboxylic acids (10 or 100 mM; pH 5). A representative experiment is shown on the left. (B) Proposed model for how acetic acid might activate TRPA1. (C) Currents evoked in HEK-293 cells expressing TRPA1 (peak magnitude at + 80 mV) in response to acetic acid at varying concentrations and pH, and to other carboxylic acids as indicated. Same color scheme as in A. Data for 0 and 10 mM of acetic acid, pH 5, were reproduced from Fig. 2 B. Representative traces of current activation by 100 mM of lactic acid (LA), pH 5, and 10 mM PA, pH 5, are shown on the left. Inset shows the structures of the acids. (D) The magnitude of the TRPA1 current plotted as a function of the change in fluorescence of the pH-sensitive dye carboxy-DFFDA. Colors correspond to the scheme in A and B. The correlation between the change in fluorescence and the magnitude of the TRPA1 current suggests that intracellular pH is the proximate stimulus that gates TRPA1 in response to extracellularly applied weak acids. Data are represented by the mean ± SEM.
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Related In: Results  -  Collection

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fig3: TRPA1 is activated by weak acids that acidify the cell cytosol. (A) Changes in emission from the pH-sensitive fluorescent probe carboxy-DFFDA in HEK-293 cells in response to acetic acid at varying concentrations (0, 0.5, 2, 10, and 100 mM at pH 5) and pH (10 mM; pH 5, 6, and 7) and to a panel of carboxylic acids (10 or 100 mM; pH 5). A representative experiment is shown on the left. (B) Proposed model for how acetic acid might activate TRPA1. (C) Currents evoked in HEK-293 cells expressing TRPA1 (peak magnitude at + 80 mV) in response to acetic acid at varying concentrations and pH, and to other carboxylic acids as indicated. Same color scheme as in A. Data for 0 and 10 mM of acetic acid, pH 5, were reproduced from Fig. 2 B. Representative traces of current activation by 100 mM of lactic acid (LA), pH 5, and 10 mM PA, pH 5, are shown on the left. Inset shows the structures of the acids. (D) The magnitude of the TRPA1 current plotted as a function of the change in fluorescence of the pH-sensitive dye carboxy-DFFDA. Colors correspond to the scheme in A and B. The correlation between the change in fluorescence and the magnitude of the TRPA1 current suggests that intracellular pH is the proximate stimulus that gates TRPA1 in response to extracellularly applied weak acids. Data are represented by the mean ± SEM.
Mentions: We previously reported that CO2 activates TRPA1 by acidifying the cell cytosol (Wang et al., 2010). Acetic acid and other carboxylic acids are well known to produce intracellular acidification and thus might activate TRPA1 through the same mechanism. Acetic acid has a pKa of 4.88, and at pH 5.0, a substantial portion of the acid (∼37%) is in the protonated membrane-permeable form. In contrast, the standard pH buffers HEPES and MES are both zwitterionic, not membrane permeable at pH 5.0, and not expected to produce intracellular acidification. We confirmed that under the conditions of our experiments, acetic acid produces intracellular acidification by measuring the response of HEK cells loaded with a pH-sensitive fluorescent probe, carboxy-DFFDA. Acidic extracellular solution containing acetic acid and not HEPES or MES produced robust intracellular acidification. Moreover, as expected, increasing the concentration of acetic acid, while holding the extracellular pH constant, produced a more robust and rapid acidification of the cell cytosol. Also, as expected, at more alkaline pH (pH 6 or pH 7), the same concentration of acetic acid produced weaker intracellular acidification (Fig. 3 A).

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