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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

Neither ASICs nor TRPV1 activities are affected by depletion of PI(3,4,5)P3.(A) CF-PTEN is rapidly recruited to the plasma membrane anchor LDR by dimerization of FRB and FKBP upon addition of rapamycin, and the PD domain of CF-PTEN specifically dephosphorylates PI(3,4,5)P3 to PI(4,5)P2. Red bar in the C-terminal tail of PTEN indicates PDZ-binding domain. (B) Confocal images of cells expressing LDR, CF-PTEN, and Btk-PH-GFP before and after the addition of rapamycin (1 μM) for 120 s (Scale bar, 5 μm) and cytosolic fluorescence intensities of CFP (blue) and GFP (green) (n = 3). The values of the Y-axis use an arbitrary unit. (C) ASIC current traces triggered by extracellular acidification in cells expressing LDR and CF (lacking PTEN) or CF-PTEN. Rapamycin (1 μM) was bath-applied for 60 s, and then normal extracellular solution was perfused for 10 s right before the second pulse to minimize possible side effects of rapamycin. Dashed line indicates the zero current level. (D) Relative current density measured for the cells in (C) (CF (n = 8), CF-PTEN (n = 7) for ASIC1a; CF (n = 8), CF-PTEN (n = 8) for ASIC2a; and CF (n = 10), CF-PTEN (n = 10) for ASIC3). Current density of each pulse was divided by that of the first pulse. There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. (E) TRPV1 currents in response to pH drop in the cells expressing LDR and CF or CF-PTEN. Rapamycin (1 μM) was co-applied for 90 s during the acid stimuli. Amiloride (300 μM) was pretreated for 30 s before the pH pulse. Black dashed line indicates the zero current level. Red dashed line indicates the point of rapamycin application. (F) Percentage of current decrease in (E) during 45 s of acidification before (grey) and after (red) rapamycin addition (n = 9 for CF; n = 10 for CF-PTEN). Data are mean ± SEM.
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pone.0122014.g004: Neither ASICs nor TRPV1 activities are affected by depletion of PI(3,4,5)P3.(A) CF-PTEN is rapidly recruited to the plasma membrane anchor LDR by dimerization of FRB and FKBP upon addition of rapamycin, and the PD domain of CF-PTEN specifically dephosphorylates PI(3,4,5)P3 to PI(4,5)P2. Red bar in the C-terminal tail of PTEN indicates PDZ-binding domain. (B) Confocal images of cells expressing LDR, CF-PTEN, and Btk-PH-GFP before and after the addition of rapamycin (1 μM) for 120 s (Scale bar, 5 μm) and cytosolic fluorescence intensities of CFP (blue) and GFP (green) (n = 3). The values of the Y-axis use an arbitrary unit. (C) ASIC current traces triggered by extracellular acidification in cells expressing LDR and CF (lacking PTEN) or CF-PTEN. Rapamycin (1 μM) was bath-applied for 60 s, and then normal extracellular solution was perfused for 10 s right before the second pulse to minimize possible side effects of rapamycin. Dashed line indicates the zero current level. (D) Relative current density measured for the cells in (C) (CF (n = 8), CF-PTEN (n = 7) for ASIC1a; CF (n = 8), CF-PTEN (n = 8) for ASIC2a; and CF (n = 10), CF-PTEN (n = 10) for ASIC3). Current density of each pulse was divided by that of the first pulse. There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. (E) TRPV1 currents in response to pH drop in the cells expressing LDR and CF or CF-PTEN. Rapamycin (1 μM) was co-applied for 90 s during the acid stimuli. Amiloride (300 μM) was pretreated for 30 s before the pH pulse. Black dashed line indicates the zero current level. Red dashed line indicates the point of rapamycin application. (F) Percentage of current decrease in (E) during 45 s of acidification before (grey) and after (red) rapamycin addition (n = 9 for CF; n = 10 for CF-PTEN). Data are mean ± SEM.

Mentions: Even though the PJ system is a powerful tool for probing the role of phosphoinositides for the function of ion channels, it has a limitation in terms of investigating the specific effect of PI(3,4,5)P3 on the channels. Therefore, we generated a novel chimeric protein to further investigate the role of PI(3,4,5)P3 in the activities of proton-sensitive ion channels. One of the tumor suppressor genes, PTEN (phosphatase and tensin homologue deleted on chromosome 10) codes a cytosolic 3-phosphatase that degrades PI(3,4,5)P3 by removing the phosphate at the D3 position of the inositol ring [46–48]. PTEN has a substrate specificity toward PI(3,4,5)P3 [49] and is composed of N-terminal phosphoinositide-binding motif (PBM) that contributes to the recruitment of PTEN to the PM, phosphatase domain (PD), C2 domain (C2) that binds to PM phosphatidylserine (PS), and C-terminal tail PDZ-binding domain [36,46–48] (Fig. 4A). We inserted the region from PD to C-terminal tail of PTEN to the C-terminus of CFP-FKBP (CF) (Fig. 4A). CFP-FKBP-PTEN (CF-PTEN) was translocated to the PM anchor LDR upon addition of 1 μM rapamycin; in turn, PI(3,4,5)P3 was depleted as shown by the specific PI(3,4,5)P3 probe, PH domain of Bruton tyrosine kinase (Btk) (Btk-PH-GFP) (Fig. 4B). The cytosolic fluorescence intensity of Btk-PH rapidly increased while that of CF-PTEN gradually declined by rapamycin addition (Fig. 4B), indicating the successful development of a novel translocatable 3-phosphatase tool.


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)

Neither ASICs nor TRPV1 activities are affected by depletion of PI(3,4,5)P3.(A) CF-PTEN is rapidly recruited to the plasma membrane anchor LDR by dimerization of FRB and FKBP upon addition of rapamycin, and the PD domain of CF-PTEN specifically dephosphorylates PI(3,4,5)P3 to PI(4,5)P2. Red bar in the C-terminal tail of PTEN indicates PDZ-binding domain. (B) Confocal images of cells expressing LDR, CF-PTEN, and Btk-PH-GFP before and after the addition of rapamycin (1 μM) for 120 s (Scale bar, 5 μm) and cytosolic fluorescence intensities of CFP (blue) and GFP (green) (n = 3). The values of the Y-axis use an arbitrary unit. (C) ASIC current traces triggered by extracellular acidification in cells expressing LDR and CF (lacking PTEN) or CF-PTEN. Rapamycin (1 μM) was bath-applied for 60 s, and then normal extracellular solution was perfused for 10 s right before the second pulse to minimize possible side effects of rapamycin. Dashed line indicates the zero current level. (D) Relative current density measured for the cells in (C) (CF (n = 8), CF-PTEN (n = 7) for ASIC1a; CF (n = 8), CF-PTEN (n = 8) for ASIC2a; and CF (n = 10), CF-PTEN (n = 10) for ASIC3). Current density of each pulse was divided by that of the first pulse. There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. (E) TRPV1 currents in response to pH drop in the cells expressing LDR and CF or CF-PTEN. Rapamycin (1 μM) was co-applied for 90 s during the acid stimuli. Amiloride (300 μM) was pretreated for 30 s before the pH pulse. Black dashed line indicates the zero current level. Red dashed line indicates the point of rapamycin application. (F) Percentage of current decrease in (E) during 45 s of acidification before (grey) and after (red) rapamycin addition (n = 9 for CF; n = 10 for CF-PTEN). Data are mean ± SEM.
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Related In: Results  -  Collection

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pone.0122014.g004: Neither ASICs nor TRPV1 activities are affected by depletion of PI(3,4,5)P3.(A) CF-PTEN is rapidly recruited to the plasma membrane anchor LDR by dimerization of FRB and FKBP upon addition of rapamycin, and the PD domain of CF-PTEN specifically dephosphorylates PI(3,4,5)P3 to PI(4,5)P2. Red bar in the C-terminal tail of PTEN indicates PDZ-binding domain. (B) Confocal images of cells expressing LDR, CF-PTEN, and Btk-PH-GFP before and after the addition of rapamycin (1 μM) for 120 s (Scale bar, 5 μm) and cytosolic fluorescence intensities of CFP (blue) and GFP (green) (n = 3). The values of the Y-axis use an arbitrary unit. (C) ASIC current traces triggered by extracellular acidification in cells expressing LDR and CF (lacking PTEN) or CF-PTEN. Rapamycin (1 μM) was bath-applied for 60 s, and then normal extracellular solution was perfused for 10 s right before the second pulse to minimize possible side effects of rapamycin. Dashed line indicates the zero current level. (D) Relative current density measured for the cells in (C) (CF (n = 8), CF-PTEN (n = 7) for ASIC1a; CF (n = 8), CF-PTEN (n = 8) for ASIC2a; and CF (n = 10), CF-PTEN (n = 10) for ASIC3). Current density of each pulse was divided by that of the first pulse. There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. (E) TRPV1 currents in response to pH drop in the cells expressing LDR and CF or CF-PTEN. Rapamycin (1 μM) was co-applied for 90 s during the acid stimuli. Amiloride (300 μM) was pretreated for 30 s before the pH pulse. Black dashed line indicates the zero current level. Red dashed line indicates the point of rapamycin application. (F) Percentage of current decrease in (E) during 45 s of acidification before (grey) and after (red) rapamycin addition (n = 9 for CF; n = 10 for CF-PTEN). Data are mean ± SEM.
Mentions: Even though the PJ system is a powerful tool for probing the role of phosphoinositides for the function of ion channels, it has a limitation in terms of investigating the specific effect of PI(3,4,5)P3 on the channels. Therefore, we generated a novel chimeric protein to further investigate the role of PI(3,4,5)P3 in the activities of proton-sensitive ion channels. One of the tumor suppressor genes, PTEN (phosphatase and tensin homologue deleted on chromosome 10) codes a cytosolic 3-phosphatase that degrades PI(3,4,5)P3 by removing the phosphate at the D3 position of the inositol ring [46–48]. PTEN has a substrate specificity toward PI(3,4,5)P3 [49] and is composed of N-terminal phosphoinositide-binding motif (PBM) that contributes to the recruitment of PTEN to the PM, phosphatase domain (PD), C2 domain (C2) that binds to PM phosphatidylserine (PS), and C-terminal tail PDZ-binding domain [36,46–48] (Fig. 4A). We inserted the region from PD to C-terminal tail of PTEN to the C-terminus of CFP-FKBP (CF) (Fig. 4A). CFP-FKBP-PTEN (CF-PTEN) was translocated to the PM anchor LDR upon addition of 1 μM rapamycin; in turn, PI(3,4,5)P3 was depleted as shown by the specific PI(3,4,5)P3 probe, PH domain of Bruton tyrosine kinase (Btk) (Btk-PH-GFP) (Fig. 4B). The cytosolic fluorescence intensity of Btk-PH rapidly increased while that of CF-PTEN gradually declined by rapamycin addition (Fig. 4B), indicating the successful development of a novel translocatable 3-phosphatase tool.

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