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Differential regulation of proton-sensitive ion channels by phospholipids: a comparative study between ASICs and TRPV1.

Kweon HJ, Yu SY, Kim DI, Suh BC - PLoS ONE (2015)

Bottom Line: We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their function.Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels.In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P2, and AA, although they have common roles as proton sensors.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Republic of Korea.

ABSTRACT
Protons are released in pain-generating pathological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons are thought to play a role in neurotransmission processes. Acid-sensing ion channels (ASICs) are typical proton sensors in the central nervous system (CNS) and the peripheral nervous system (PNS). In addition to ASICs, capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channels can also mediate proton-mediated pain signaling. In spite of their importance in perception of pH fluctuations, the regulatory mechanisms of these proton-sensitive ion channels still need to be further investigated. Here, we compared regulation of ASICs and TRPV1 by membrane phosphoinositides, which are general cofactors of many receptors and ion channels. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P2. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P3 in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P2, and AA, although they have common roles as proton sensors. Further investigation about the complementary roles and respective contributions of ASICs and TRPV1 in proton-mediated signaling is necessary.

No MeSH data available.


Related in: MedlinePlus

Potentiation of ASICs by AA.(A) ASIC current traces activated by rapid extracellular pH changes. AA (10 μM) was bath-applied for 20 s before the second pulse. Dashed line indicates the zero current level. (B) Relative current density was measured for the cells expressing ASIC1a (n = 5 for DMSO; n = 6 for AA), ASIC2a (n = 5 for DMSO; n = 10 for AA), and ASIC3 (n = 5 for DMSO; n = 12 for AA). Current density of each pulse was divided by that of the first pulse. * P < 0.05 and ** P < 0.01, with two-way ANOVA followed by Bonferroni post-hoc test and student’s t-test. (C) Dose-dependent relative current density of ASIC1a (blue) (n = 5–20), ASIC2a (green) (n = 5–25), and ASIC3 (red) (n = 5–23). (D) ASIC1a and ASIC3 currents were inhibited by preincubation of cells with pH 7.4 solution containing amiloride (300 μM) for 20 s before the second pulse. In the case of ASIC2a, 600 μM of amiloride was applied for 30 s before and during the second pulse. (E) Percentage of inhibition by amiloride in the absence (grey) or the presence (yellow) of AA (AMI (n = 7) and AA+AMI (n = 4) for ASIC1a; AMI (n = 4) and AA+AMI (n = 3) for ASIC2a; and AMI (n = 6) and AA+AMI (n = 6) for ASIC3). (F) The potentiating effect of AA (10 μM) on ASIC currents was inhibited by amiloride. Data are mean ± SEM.
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pone.0122014.g005: Potentiation of ASICs by AA.(A) ASIC current traces activated by rapid extracellular pH changes. AA (10 μM) was bath-applied for 20 s before the second pulse. Dashed line indicates the zero current level. (B) Relative current density was measured for the cells expressing ASIC1a (n = 5 for DMSO; n = 6 for AA), ASIC2a (n = 5 for DMSO; n = 10 for AA), and ASIC3 (n = 5 for DMSO; n = 12 for AA). Current density of each pulse was divided by that of the first pulse. * P < 0.05 and ** P < 0.01, with two-way ANOVA followed by Bonferroni post-hoc test and student’s t-test. (C) Dose-dependent relative current density of ASIC1a (blue) (n = 5–20), ASIC2a (green) (n = 5–25), and ASIC3 (red) (n = 5–23). (D) ASIC1a and ASIC3 currents were inhibited by preincubation of cells with pH 7.4 solution containing amiloride (300 μM) for 20 s before the second pulse. In the case of ASIC2a, 600 μM of amiloride was applied for 30 s before and during the second pulse. (E) Percentage of inhibition by amiloride in the absence (grey) or the presence (yellow) of AA (AMI (n = 7) and AA+AMI (n = 4) for ASIC1a; AMI (n = 4) and AA+AMI (n = 3) for ASIC2a; and AMI (n = 6) and AA+AMI (n = 6) for ASIC3). (F) The potentiating effect of AA (10 μM) on ASIC currents was inhibited by amiloride. Data are mean ± SEM.

Mentions: When the extracellular solution containing 10 μM of AA was perfused for 20 s right before the second pH pulse, the peak current density of the second pulse in cells transiently expressing homomeric ASIC1a channels was increased by 81 ± 29% (n = 6) compared to that of the first pulse (Fig. 5A-B). On the other hand, the peak current density of the second pulse was slightly decreased in the control group (Fig. 5B). We observed that the potentiating effect of AA is reversible, and the peak current density was recovered to the initial level after washout of AA (Fig. 5A-B). In the cells expressing ASIC2a homomeric channels, the peak current density of the second pulse was reversibly increased by 103 ± 30% (n = 10) compared to that of the first pulse, whereas, in the control group, the difference in the current density between the first and the second pulses was negligible (Fig. 5A-B). Similarly, the peak current density of the second pulse in cells expressing ASIC3 homomeric channels was reversibly increased by 133 ± 33% (n = 12) compared to that of the first pulse (Fig. 5A-B). AA increased the respective ASIC currents in a dose-dependent manner (Fig. 5C). ASIC1a, ASIC2a, and ASIC3 homomeric channels displayed similar dose-dependent curves.


Differential regulation of proton-sensitive ion channels by phospholipids: a comparative study between ASICs and TRPV1.

Kweon HJ, Yu SY, Kim DI, Suh BC - PLoS ONE (2015)

Potentiation of ASICs by AA.(A) ASIC current traces activated by rapid extracellular pH changes. AA (10 μM) was bath-applied for 20 s before the second pulse. Dashed line indicates the zero current level. (B) Relative current density was measured for the cells expressing ASIC1a (n = 5 for DMSO; n = 6 for AA), ASIC2a (n = 5 for DMSO; n = 10 for AA), and ASIC3 (n = 5 for DMSO; n = 12 for AA). Current density of each pulse was divided by that of the first pulse. * P < 0.05 and ** P < 0.01, with two-way ANOVA followed by Bonferroni post-hoc test and student’s t-test. (C) Dose-dependent relative current density of ASIC1a (blue) (n = 5–20), ASIC2a (green) (n = 5–25), and ASIC3 (red) (n = 5–23). (D) ASIC1a and ASIC3 currents were inhibited by preincubation of cells with pH 7.4 solution containing amiloride (300 μM) for 20 s before the second pulse. In the case of ASIC2a, 600 μM of amiloride was applied for 30 s before and during the second pulse. (E) Percentage of inhibition by amiloride in the absence (grey) or the presence (yellow) of AA (AMI (n = 7) and AA+AMI (n = 4) for ASIC1a; AMI (n = 4) and AA+AMI (n = 3) for ASIC2a; and AMI (n = 6) and AA+AMI (n = 6) for ASIC3). (F) The potentiating effect of AA (10 μM) on ASIC currents was inhibited by amiloride. Data are mean ± SEM.
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Related In: Results  -  Collection

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pone.0122014.g005: Potentiation of ASICs by AA.(A) ASIC current traces activated by rapid extracellular pH changes. AA (10 μM) was bath-applied for 20 s before the second pulse. Dashed line indicates the zero current level. (B) Relative current density was measured for the cells expressing ASIC1a (n = 5 for DMSO; n = 6 for AA), ASIC2a (n = 5 for DMSO; n = 10 for AA), and ASIC3 (n = 5 for DMSO; n = 12 for AA). Current density of each pulse was divided by that of the first pulse. * P < 0.05 and ** P < 0.01, with two-way ANOVA followed by Bonferroni post-hoc test and student’s t-test. (C) Dose-dependent relative current density of ASIC1a (blue) (n = 5–20), ASIC2a (green) (n = 5–25), and ASIC3 (red) (n = 5–23). (D) ASIC1a and ASIC3 currents were inhibited by preincubation of cells with pH 7.4 solution containing amiloride (300 μM) for 20 s before the second pulse. In the case of ASIC2a, 600 μM of amiloride was applied for 30 s before and during the second pulse. (E) Percentage of inhibition by amiloride in the absence (grey) or the presence (yellow) of AA (AMI (n = 7) and AA+AMI (n = 4) for ASIC1a; AMI (n = 4) and AA+AMI (n = 3) for ASIC2a; and AMI (n = 6) and AA+AMI (n = 6) for ASIC3). (F) The potentiating effect of AA (10 μM) on ASIC currents was inhibited by amiloride. Data are mean ± SEM.
Mentions: When the extracellular solution containing 10 μM of AA was perfused for 20 s right before the second pH pulse, the peak current density of the second pulse in cells transiently expressing homomeric ASIC1a channels was increased by 81 ± 29% (n = 6) compared to that of the first pulse (Fig. 5A-B). On the other hand, the peak current density of the second pulse was slightly decreased in the control group (Fig. 5B). We observed that the potentiating effect of AA is reversible, and the peak current density was recovered to the initial level after washout of AA (Fig. 5A-B). In the cells expressing ASIC2a homomeric channels, the peak current density of the second pulse was reversibly increased by 103 ± 30% (n = 10) compared to that of the first pulse, whereas, in the control group, the difference in the current density between the first and the second pulses was negligible (Fig. 5A-B). Similarly, the peak current density of the second pulse in cells expressing ASIC3 homomeric channels was reversibly increased by 133 ± 33% (n = 12) compared to that of the first pulse (Fig. 5A-B). AA increased the respective ASIC currents in a dose-dependent manner (Fig. 5C). ASIC1a, ASIC2a, and ASIC3 homomeric channels displayed similar dose-dependent curves.

Bottom Line: We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their function.Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels.In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P2, and AA, although they have common roles as proton sensors.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Republic of Korea.

ABSTRACT
Protons are released in pain-generating pathological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons are thought to play a role in neurotransmission processes. Acid-sensing ion channels (ASICs) are typical proton sensors in the central nervous system (CNS) and the peripheral nervous system (PNS). In addition to ASICs, capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channels can also mediate proton-mediated pain signaling. In spite of their importance in perception of pH fluctuations, the regulatory mechanisms of these proton-sensitive ion channels still need to be further investigated. Here, we compared regulation of ASICs and TRPV1 by membrane phosphoinositides, which are general cofactors of many receptors and ion channels. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P2. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P3 in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P2, and AA, although they have common roles as proton sensors. Further investigation about the complementary roles and respective contributions of ASICs and TRPV1 in proton-mediated signaling is necessary.

No MeSH data available.


Related in: MedlinePlus