Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux

Dino Luethi; Julian Maier; Deborah Rudin; Dániel Szöllősi; Thomas J.F. Angenoorth; Stevan Stankovic; Matthias Schittmayer; Isabella Burger; Jae Won Yang; Kathrin Jaentsch; Marion Holy; Anand Kant Das; Mario Brameshuber; Gisela Andrea Camacho-Hernandez; Andrea Casiraghi; Amy Hauck Newman; Oliver Kudlacek; Ruth Birner-Gruenberger; Thomas Stockner; Gerhard J. Schütz; Harald H. Sitte

doi:10.1038/s42003-022-04210-1

Phosphatidylinositol 4,5-bisphosphate (PIP₂) facilitates norepinephrine transporter dimerization and modulates substrate efflux

Dino Luethi, Julian Maier, Deborah Rudin, Dániel Szöllősi, Thomas J.F. Angenoorth, Stevan Stankovic, Matthias Schittmayer, Isabella Burger, Jae Won Yang, Kathrin Jaentsch, Marion Holy, Anand Kant Das, Mario Brameshuber, Gisela Andrea Camacho-Hernandez, Andrea Casiraghi, Amy Hauck Newman, Oliver Kudlacek, Ruth Birner-Gruenberger, Thomas Stockner, Gerhard J. SchützHarald H. Sitte

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP₂) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP₂ depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP₂ binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP₂ depletion; this indicates that PIP₂ interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP₂ signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP₂ directly regulates NET organization and function.

Original language	English (US)
Article number	1259
Journal	Communications Biology
Volume	5
Issue number	1
DOIs	https://doi.org/10.1038/s42003-022-04210-1
State	Published - Dec 2022
Externally published	Yes

ASJC Scopus subject areas

Medicine (miscellaneous)
General Biochemistry, Genetics and Molecular Biology
General Agricultural and Biological Sciences

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Luethi, D., Maier, J., Rudin, D., Szöllősi, D., Angenoorth, T. J. F., Stankovic, S., Schittmayer, M., Burger, I., Yang, J. W., Jaentsch, K., Holy, M., Das, A. K., Brameshuber, M., Camacho-Hernandez, G. A., Casiraghi, A., Newman, A. H., Kudlacek, O., Birner-Gruenberger, R., Stockner, T., ... Sitte, H. H. (2022). Phosphatidylinositol 4,5-bisphosphate (PIP₂) facilitates norepinephrine transporter dimerization and modulates substrate efflux. Communications Biology, 5(1), Article 1259. https://doi.org/10.1038/s42003-022-04210-1

Luethi, D, Maier, J, Rudin, D, Szöllősi, D, Angenoorth, TJF, Stankovic, S, Schittmayer, M, Burger, I, Yang, JW, Jaentsch, K, Holy, M, Das, AK, Brameshuber, M, Camacho-Hernandez, GA, Casiraghi, A, Newman, AH, Kudlacek, O, Birner-Gruenberger, R, Stockner, T, Schütz, GJ & Sitte, HH 2022, 'Phosphatidylinositol 4,5-bisphosphate (PIP₂) facilitates norepinephrine transporter dimerization and modulates substrate efflux', Communications Biology, vol. 5, no. 1, 1259. https://doi.org/10.1038/s42003-022-04210-1

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title = "Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux",

abstract = "The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.",

author = "Dino Luethi and Julian Maier and Deborah Rudin and D{\'a}niel Sz{\"o}ll{\H o}si and Angenoorth, {Thomas J.F.} and Stevan Stankovic and Matthias Schittmayer and Isabella Burger and Yang, {Jae Won} and Kathrin Jaentsch and Marion Holy and Das, {Anand Kant} and Mario Brameshuber and Camacho-Hernandez, {Gisela Andrea} and Andrea Casiraghi and Newman, {Amy Hauck} and Oliver Kudlacek and Ruth Birner-Gruenberger and Thomas Stockner and Sch{\"u}tz, {Gerhard J.} and Sitte, {Harald H.}",

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doi = "10.1038/s42003-022-04210-1",

language = "English (US)",

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AU - Luethi, Dino

AU - Maier, Julian

AU - Rudin, Deborah

AU - Szöllősi, Dániel

AU - Angenoorth, Thomas J.F.

AU - Stankovic, Stevan

AU - Schittmayer, Matthias

AU - Burger, Isabella

AU - Yang, Jae Won

AU - Jaentsch, Kathrin

AU - Holy, Marion

AU - Das, Anand Kant

AU - Brameshuber, Mario

AU - Camacho-Hernandez, Gisela Andrea

AU - Casiraghi, Andrea

AU - Newman, Amy Hauck

AU - Kudlacek, Oliver

AU - Birner-Gruenberger, Ruth

AU - Stockner, Thomas

AU - Schütz, Gerhard J.

AU - Sitte, Harald H.

PY - 2022/12

Y1 - 2022/12

N2 - The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

AB - The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

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Phosphatidylinositol 4,5-bisphosphate (PIP₂) facilitates norepinephrine transporter dimerization and modulates substrate efflux

Abstract

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