Methods to study phosphoinositide regulation of ion channels

Yudin, Yevgen; Liu, Luyu; Nagwekar, Janhavi; Rohács, Tibor

doi:10.1016/bs.mie.2021.01.025

Cited by 6 publications

(5 citation statements)

References 132 publications

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“…We showed earlier that at 35 nM, a concentration that inhibits PI3K, wortmannin did not inhibit TRPM3 (21), indicating that TRPM3 inhibition by 35 μM wortmannin is caused by inhibition of PI4K, not PI3K. Mutating a PI(4,5)P2 interacting residue is expected to increase inhibition by high concentrations of wortmannin (39). We mutated the TRP domain positively charged residues to Q, as equivalent mutations in TRPM8 were shown to be functional, and affect PI(4,5)P2 interactions (36).…”

Section: Resultsmentioning

confidence: 92%

“…3A,B). Wortmannin at this concentration inhibits phosphatidylinositol 4-kinases (PI4K), and has been used to inhibit the activity of PI(4,5)P2 dependent ion channels (39). We showed earlier that at 35 nM, a concentration that inhibits PI3K, wortmannin did not inhibit TRPM3 (21), indicating that TRPM3 inhibition by 35 μM wortmannin is caused by inhibition of PI4K, not PI3K.…”

Section: Resultsmentioning

confidence: 99%

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Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Zhao

Carnevale²,

Gabrielle³

et al. 2022

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Zhao

Carnevale²,

Gabrielle³

et al. 2022

Journal of Biological Chemistry

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“…3B,C ). Wortmannin at this concentration inhibits phosphatidylinositol 4-kinases (PI4K), and has been used to inhibit the activity of PI(4,5)P 2 dependent ion channels (39). Mutating a PI(4,5)P 2 interacting residue is expected to increase inhibition by wortmannin (39).…”

Section: Resultsmentioning

confidence: 99%

“…Wortmannin at this concentration inhibits phosphatidylinositol 4-kinases (PI4K), and has been used to inhibit the activity of PI(4,5)P 2 dependent ion channels (39). Mutating a PI(4,5)P 2 interacting residue is expected to increase inhibition by wortmannin (39). We mutated the TRP domain positively charged residues to Q, as equivalent mutations in TRPM8 were shown to be functional, and affect PI(4,5)P 2 interactions (36).…”

Section: Resultsmentioning

confidence: 99%

Computational and functional characterization of the PI(4,5)P₂ binding site of the TRPM3 ion channel

Zhao

Carnevale

Gianti

et al. 2022

Preprint

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Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but the molecular mechanism of activation by PI(4,5)P2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P2 via molecular modeling. We identified putative contact residues for PI(4,5)P2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP-domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P2 levels by phosphatidylinositol 4-kinase inhibition. Changes in ligand-binding affinities via MM/GBSA showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2 interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate (PregS), pointing to an allosteric interaction between PI(4,5)P2 and PregS. Mutating residues in the PI(4,5)P2 binding site in TRPM8 had similar effects, increased sensitivity to PI(4,5)P2 depletion, and reduced sensitivity to menthol. Mutation of most PI(4,5)P2 interacting residues in TRPM3 also increased sensitivity to inhibition by Gβγ, indicating allosteric interaction between Gβγ and PI(4,5)P2. Disease-associated gain of function TRPM3 mutations on the other hand, resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors of TRPM3, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8.

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“…PIP 2 is a necessary cofactor of TRPM7 and other TRP channels (reviewed in Suh and Hille, 2008 ; Rohacs, 2014 ). The voltage-sensitive lipid phosphatase (VSP) from Ciona is a convenient tool for depleting phosphoinositides in intact cells ( Yudin et al, 2021 ). We showed previously that depletion of PIP 2 by overexpressing VSP mimicked inhibition of TRPM7 by cytosolic cations ( Zhelay et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

NSAIDs Naproxen, Ibuprofen, Salicylate, and Aspirin Inhibit TRPM7 Channels by Cytosolic Acidification

et al. 2021

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Non-steroidal anti-inflammatory drugs (NSAIDs) are used for relieving pain and inflammation accompanying numerous disease states. The primary therapeutic mechanism of these widely used drugs is the inhibition of cyclooxygenase 1 and 2 (COX1, 2) enzymes that catalyze the conversion of arachidonic acid into prostaglandins. At higher doses, NSAIDs are used for prevention of certain types of cancer and as experimental treatments for Alzheimer’s disease. In the immune system, various NSAIDs have been reported to influence neutrophil function and lymphocyte proliferation, and affect ion channels and cellular calcium homeostasis. Transient receptor potential melastatin 7 (TRPM7) cation channels are highly expressed in T lymphocytes and are inhibited by Mg2+, acidic pH, and polyamines. Here, we report a novel effect of naproxen, ibuprofen, salicylate, and acetylsalicylate on TRPM7. At concentrations of 3–30mM, they reversibly inhibited TRPM7 channel currents. By measuring intracellular pH with the ratiometric indicator BCECF, we found that at 300μM to 30mM, these NSAIDs reversibly acidified the cytoplasm in a concentration-dependent manner, and propose that TRPM7 channel inhibition is a consequence of cytosolic acidification, rather than direct. NSAID inhibition of TRPM7 channels was slow, voltage-independent, and displayed use-dependence, increasing in potency upon repeated drug applications. The extent of channel inhibition by salicylate strongly depended on cellular PI(4,5)P2 levels, as revealed when this phospholipid was depleted with voltage-sensitive lipid phosphatase (VSP). Salicylate inhibited heterologously expressed wildtype TRPM7 channels but not the S1107R variant, which is insensitive to cytosolic pH, Mg2+, and PI(4,5)P2 depletion. NSAID-induced acidification was also observed in Schneider 2 cells from Drosophila, an organism that lacks orthologous COX genes, suggesting that this effect is unrelated to COX enzyme activity. A 24-h exposure to 300μM–10mM naproxen resulted in a concentration-dependent reduction in cell viability. In addition to TRPM7, the described NSAID effect would be expected to apply to other ion channels and transporters sensitive to intracellular pH.

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Methods to study phosphoinositide regulation of ion channels

Cited by 6 publications

References 132 publications

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Computational and functional characterization of the PI(4,5)P₂ binding site of the TRPM3 ion channel

NSAIDs Naproxen, Ibuprofen, Salicylate, and Aspirin Inhibit TRPM7 Channels by Cytosolic Acidification

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Methods to study phosphoinositide regulation of ion channels

Cited by 6 publications

References 132 publications

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Computational and functional characterization of the PI(4,5)P2 binding site of the TRPM3 ion channel

NSAIDs Naproxen, Ibuprofen, Salicylate, and Aspirin Inhibit TRPM7 Channels by Cytosolic Acidification

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Computational and functional characterization of the PI(4,5)P₂ binding site of the TRPM3 ion channel