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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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Acetic acid activates TRPA1 but not TRPV1. Whole cell currents evoked in response to acids from untransfected HEK-293 cells or HEK-293 cells transfected with YFP-rTRPA1 or rTRPV1 as indicated. (A) 10 mM of acetic acid (HOAc) titrated to pH 5, but not 10 mM MES, pH 5, or 10 mM HEPES, pH 5, activated a large, outwardly rectifying current in TRPA1-expressing cells (right). Current–voltage relationship obtained from ramp depolarization (1V/s) at the time indicated shows reversal of the current near 0 mV and mild outward rectification. Small currents were evoked in untransfected cells in response to pH 5.0 solution buffered with MES, HEPES, or acetic acid (left). (B) Average data from experiments as in A show that the activation of TRPA1 by acetic acid is conserved in humans and rodents. Significance (compared with activation of untransfected cells by acetic acid) was determined with the one-tailed Student’s t test (n = 4–7). Representative traces for responses from mouse or human transfected cells are shown on the right. (C) 10 mM MES, pH 5.0, activated a large TRPV1 current, which was inhibited by subsequent application of 10 mM of acetic acid, pH 5. (D) Average data (n = 10) from experiments as in C, where current decay was measured during two 10-s windows immediately before and after acetic acid application. Significance was determined by the Wilcoxon signed-rank test. Average data are represented by the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig2: Acetic acid activates TRPA1 but not TRPV1. Whole cell currents evoked in response to acids from untransfected HEK-293 cells or HEK-293 cells transfected with YFP-rTRPA1 or rTRPV1 as indicated. (A) 10 mM of acetic acid (HOAc) titrated to pH 5, but not 10 mM MES, pH 5, or 10 mM HEPES, pH 5, activated a large, outwardly rectifying current in TRPA1-expressing cells (right). Current–voltage relationship obtained from ramp depolarization (1V/s) at the time indicated shows reversal of the current near 0 mV and mild outward rectification. Small currents were evoked in untransfected cells in response to pH 5.0 solution buffered with MES, HEPES, or acetic acid (left). (B) Average data from experiments as in A show that the activation of TRPA1 by acetic acid is conserved in humans and rodents. Significance (compared with activation of untransfected cells by acetic acid) was determined with the one-tailed Student’s t test (n = 4–7). Representative traces for responses from mouse or human transfected cells are shown on the right. (C) 10 mM MES, pH 5.0, activated a large TRPV1 current, which was inhibited by subsequent application of 10 mM of acetic acid, pH 5. (D) Average data (n = 10) from experiments as in C, where current decay was measured during two 10-s windows immediately before and after acetic acid application. Significance was determined by the Wilcoxon signed-rank test. Average data are represented by the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: Many weak acids, in the protonated form, can diffuse across cell membrane and cause intracellular acidification. Our previous results showed that the ion channel TRPA1, which is expressed in a subset of trigeminal neurons, is activated by intracellular protons (Wang et al., 2010), making it a possible sensor for weak acids in the nociceptive pathway. To determine whether TRPA1 could be activated by weak acids, we expressed rTRPA1 in HEK-293 cells and measured responses to applied acids with whole cell patch clamp electrophysiology. TRPA1 currents were strongly and rapidly activated by acetic acid (HOAc), pH 5.0 (Fig. 2, A and B; I+80 = 4,642 pA ± 1,276; n = 7). The response to acetic acid, pH 5.0, was significantly larger than the response to a solution of pH 5.0 that did not contain acetic acid, indicating that it could not be attributed to the effects of protons binding on the outside of the cell. This current was not observed in untransfected HEK-293 cells, where instead acetic acid evoked a rapidly inactivating Na+-selective ASIC current and a small noninactivating Cl− current (Fig. 2, A and B). The acetic acid–activated current in TRPA1-expressing HEK-293 cells reversed at −3.6 ± 1.0 mV (n = 7), as expected for a nonselective cation channel, and showed mild outward rectification (−I+80/I−80 = 3.3 ± 0.4; n = 7; Fig. 2 A). As these experiments were performed with rTRPA1 fused to YFP, we confirmed that acetic acid also activated an rTRPA1 construct not fused to YFP (Fig. 2 B). Acetic acid also activated large currents in a subset of HEK cells transfected with human TRPA1 (hTRPA1) or mouse TRPA1 (mTRPA1; Fig. 2 B), indicating that activation of the channel by acetic acid is a conserved property of the channel and may underlie irritant effects of weak acids in humans as well as in rodents.


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)

Acetic acid activates TRPA1 but not TRPV1. Whole cell currents evoked in response to acids from untransfected HEK-293 cells or HEK-293 cells transfected with YFP-rTRPA1 or rTRPV1 as indicated. (A) 10 mM of acetic acid (HOAc) titrated to pH 5, but not 10 mM MES, pH 5, or 10 mM HEPES, pH 5, activated a large, outwardly rectifying current in TRPA1-expressing cells (right). Current–voltage relationship obtained from ramp depolarization (1V/s) at the time indicated shows reversal of the current near 0 mV and mild outward rectification. Small currents were evoked in untransfected cells in response to pH 5.0 solution buffered with MES, HEPES, or acetic acid (left). (B) Average data from experiments as in A show that the activation of TRPA1 by acetic acid is conserved in humans and rodents. Significance (compared with activation of untransfected cells by acetic acid) was determined with the one-tailed Student’s t test (n = 4–7). Representative traces for responses from mouse or human transfected cells are shown on the right. (C) 10 mM MES, pH 5.0, activated a large TRPV1 current, which was inhibited by subsequent application of 10 mM of acetic acid, pH 5. (D) Average data (n = 10) from experiments as in C, where current decay was measured during two 10-s windows immediately before and after acetic acid application. Significance was determined by the Wilcoxon signed-rank test. Average data are represented by the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105510&req=5

fig2: Acetic acid activates TRPA1 but not TRPV1. Whole cell currents evoked in response to acids from untransfected HEK-293 cells or HEK-293 cells transfected with YFP-rTRPA1 or rTRPV1 as indicated. (A) 10 mM of acetic acid (HOAc) titrated to pH 5, but not 10 mM MES, pH 5, or 10 mM HEPES, pH 5, activated a large, outwardly rectifying current in TRPA1-expressing cells (right). Current–voltage relationship obtained from ramp depolarization (1V/s) at the time indicated shows reversal of the current near 0 mV and mild outward rectification. Small currents were evoked in untransfected cells in response to pH 5.0 solution buffered with MES, HEPES, or acetic acid (left). (B) Average data from experiments as in A show that the activation of TRPA1 by acetic acid is conserved in humans and rodents. Significance (compared with activation of untransfected cells by acetic acid) was determined with the one-tailed Student’s t test (n = 4–7). Representative traces for responses from mouse or human transfected cells are shown on the right. (C) 10 mM MES, pH 5.0, activated a large TRPV1 current, which was inhibited by subsequent application of 10 mM of acetic acid, pH 5. (D) Average data (n = 10) from experiments as in C, where current decay was measured during two 10-s windows immediately before and after acetic acid application. Significance was determined by the Wilcoxon signed-rank test. Average data are represented by the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: Many weak acids, in the protonated form, can diffuse across cell membrane and cause intracellular acidification. Our previous results showed that the ion channel TRPA1, which is expressed in a subset of trigeminal neurons, is activated by intracellular protons (Wang et al., 2010), making it a possible sensor for weak acids in the nociceptive pathway. To determine whether TRPA1 could be activated by weak acids, we expressed rTRPA1 in HEK-293 cells and measured responses to applied acids with whole cell patch clamp electrophysiology. TRPA1 currents were strongly and rapidly activated by acetic acid (HOAc), pH 5.0 (Fig. 2, A and B; I+80 = 4,642 pA ± 1,276; n = 7). The response to acetic acid, pH 5.0, was significantly larger than the response to a solution of pH 5.0 that did not contain acetic acid, indicating that it could not be attributed to the effects of protons binding on the outside of the cell. This current was not observed in untransfected HEK-293 cells, where instead acetic acid evoked a rapidly inactivating Na+-selective ASIC current and a small noninactivating Cl− current (Fig. 2, A and B). The acetic acid–activated current in TRPA1-expressing HEK-293 cells reversed at −3.6 ± 1.0 mV (n = 7), as expected for a nonselective cation channel, and showed mild outward rectification (−I+80/I−80 = 3.3 ± 0.4; n = 7; Fig. 2 A). As these experiments were performed with rTRPA1 fused to YFP, we confirmed that acetic acid also activated an rTRPA1 construct not fused to YFP (Fig. 2 B). Acetic acid also activated large currents in a subset of HEK cells transfected with human TRPA1 (hTRPA1) or mouse TRPA1 (mTRPA1; Fig. 2 B), indicating that activation of the channel by acetic acid is a conserved property of the channel and may underlie irritant effects of weak acids in humans as well as in rodents.

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