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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

Homomeric ASIC currents are insensitive to PI(4)P and PI(4,5)P2.(A) ASIC1a currents evoked by repetitive rapid extracellular pH change from 7.4 to 6.0 for 10 s with time intervals of 120 s in cells expressing LDR and either PJ-Dead, PJ-Sac, INPP5E, or PJ. 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. (B) Relative current density measured for the cells in (A) (n = 6, respectively). 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. (C) ASIC2a or ASIC3 current traces evoked by pH drop to 4.5 or 6.0 for 10 s. (D) Relative current density measured for the cells in (C) (n = 6, respectively). There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SEM.
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pone.0122014.g002: Homomeric ASIC currents are insensitive to PI(4)P and PI(4,5)P2.(A) ASIC1a currents evoked by repetitive rapid extracellular pH change from 7.4 to 6.0 for 10 s with time intervals of 120 s in cells expressing LDR and either PJ-Dead, PJ-Sac, INPP5E, or PJ. 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. (B) Relative current density measured for the cells in (A) (n = 6, respectively). 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. (C) ASIC2a or ASIC3 current traces evoked by pH drop to 4.5 or 6.0 for 10 s. (D) Relative current density measured for the cells in (C) (n = 6, respectively). There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SEM.

Mentions: Next, we examined whether ASICs also require phosphoinositides for their function as TRPV1 channels. ENaC belongs to the same superfamily of ion channels as ASICs and is known to be regulated by PM PI(4,5)P2 and PI(3,4,5)P3 [20–22]. That means, ASICs could have sensitivities toward phosphoinositides. To test this, we employed the PJ system and observed the activities of homomeric and heteromeric ASICs. The cells transiently expressing each GFP-tagged ASIC subunit (ASIC1a, or ASIC2a, or ASIC3) and respective PJ systems were repetitively activated by extracellular acidification from pH 7.4 to pH 6.0 (ASIC1a or ASIC3) or to pH 4.5 (ASIC2a) for 10 s with 2 min time intervals. As previously reported, GFP fusion to the C-terminus of ASIC subunit did not affect the electrophysiological properties of wild-type ASIC currents expressed in tsA201 cells [40]. For recruiting the interested enzyme to the PM, 1 μM of rapamycin was perfused for 60 s before the second pH pulse. To minimize possible side effects of rapamycin, normal extracellular solution was perfused right after the application of rapamycin for 10 s just before the second pH pulse. First, we confirmed that, as a control, the recruitment of PJ-Dead to the PM anchor does not affect the repetitive proton-activated ASIC1a currents except for tachyphylaxis (reduction in current amplitude with repeated stimulation), a unique property of homomeric ASIC1a channels [41] (Fig. 2A-B). Then, we observed that translocation of PJ-Sac or INPP5E to the PM to specifically deplete PM PI(4)P or PI(4,5)P2, respectively, had no effects on the relative current density of homomeric ASIC1a channels (Fig. 2A-B). Simultaneous depletion of both PI(4)P and PI(4,5)P2 by PJ also had no significant effect on the successively triggered ASIC1a currents (Fig. 2A-B). We found that neither ASIC2a nor ASIC3 homomeric channels were affected by the recruitment of PJ to the PM (Fig. 2C-D), allowing us to conclude that unlike proton-sensitive TRPV1 channels, the activities of homomeric ASICs are independent of PM PI(4)P and PI(4,5)P2.


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)

Homomeric ASIC currents are insensitive to PI(4)P and PI(4,5)P2.(A) ASIC1a currents evoked by repetitive rapid extracellular pH change from 7.4 to 6.0 for 10 s with time intervals of 120 s in cells expressing LDR and either PJ-Dead, PJ-Sac, INPP5E, or PJ. 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. (B) Relative current density measured for the cells in (A) (n = 6, respectively). 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. (C) ASIC2a or ASIC3 current traces evoked by pH drop to 4.5 or 6.0 for 10 s. (D) Relative current density measured for the cells in (C) (n = 6, respectively). There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362947&req=5

pone.0122014.g002: Homomeric ASIC currents are insensitive to PI(4)P and PI(4,5)P2.(A) ASIC1a currents evoked by repetitive rapid extracellular pH change from 7.4 to 6.0 for 10 s with time intervals of 120 s in cells expressing LDR and either PJ-Dead, PJ-Sac, INPP5E, or PJ. 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. (B) Relative current density measured for the cells in (A) (n = 6, respectively). 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. (C) ASIC2a or ASIC3 current traces evoked by pH drop to 4.5 or 6.0 for 10 s. (D) Relative current density measured for the cells in (C) (n = 6, respectively). There is no statistical significance with two-way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SEM.
Mentions: Next, we examined whether ASICs also require phosphoinositides for their function as TRPV1 channels. ENaC belongs to the same superfamily of ion channels as ASICs and is known to be regulated by PM PI(4,5)P2 and PI(3,4,5)P3 [20–22]. That means, ASICs could have sensitivities toward phosphoinositides. To test this, we employed the PJ system and observed the activities of homomeric and heteromeric ASICs. The cells transiently expressing each GFP-tagged ASIC subunit (ASIC1a, or ASIC2a, or ASIC3) and respective PJ systems were repetitively activated by extracellular acidification from pH 7.4 to pH 6.0 (ASIC1a or ASIC3) or to pH 4.5 (ASIC2a) for 10 s with 2 min time intervals. As previously reported, GFP fusion to the C-terminus of ASIC subunit did not affect the electrophysiological properties of wild-type ASIC currents expressed in tsA201 cells [40]. For recruiting the interested enzyme to the PM, 1 μM of rapamycin was perfused for 60 s before the second pH pulse. To minimize possible side effects of rapamycin, normal extracellular solution was perfused right after the application of rapamycin for 10 s just before the second pH pulse. First, we confirmed that, as a control, the recruitment of PJ-Dead to the PM anchor does not affect the repetitive proton-activated ASIC1a currents except for tachyphylaxis (reduction in current amplitude with repeated stimulation), a unique property of homomeric ASIC1a channels [41] (Fig. 2A-B). Then, we observed that translocation of PJ-Sac or INPP5E to the PM to specifically deplete PM PI(4)P or PI(4,5)P2, respectively, had no effects on the relative current density of homomeric ASIC1a channels (Fig. 2A-B). Simultaneous depletion of both PI(4)P and PI(4,5)P2 by PJ also had no significant effect on the successively triggered ASIC1a currents (Fig. 2A-B). We found that neither ASIC2a nor ASIC3 homomeric channels were affected by the recruitment of PJ to the PM (Fig. 2C-D), allowing us to conclude that unlike proton-sensitive TRPV1 channels, the activities of homomeric ASICs are independent of PM PI(4)P and PI(4,5)P2.

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