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Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility
The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P(2) are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P(2). Yet, measur...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2015
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472987/ https://www.ncbi.nlm.nih.gov/pubmed/26150791 http://dx.doi.org/10.3389/fphar.2015.00127 |
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author | Rjasanow, Alexandra Leitner, Michael G. Thallmair, Veronika Halaszovich, Christian R. Oliver, Dominik |
author_facet | Rjasanow, Alexandra Leitner, Michael G. Thallmair, Veronika Halaszovich, Christian R. Oliver, Dominik |
author_sort | Rjasanow, Alexandra |
collection | PubMed |
description | The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P(2) are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P(2). Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P(2) to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P(2). Because cellular PI(4,5)P(2) is resynthesized rapidly, steady state PI(4,5)P(2) changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P(2) with Ci-VSP allows for the quantification of relative PI(4,5)P(2) affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K(+) channels to Ci-VSP allowed for comparison of PI(4,5)P(2) affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P(2) depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P(2) and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P(2) affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P(2). While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P(2) affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P(2) that differ in their accessibility to PLC and VSPs. |
format | Online Article Text |
id | pubmed-4472987 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-44729872015-07-06 Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility Rjasanow, Alexandra Leitner, Michael G. Thallmair, Veronika Halaszovich, Christian R. Oliver, Dominik Front Pharmacol Pharmacology The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P(2) are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P(2). Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P(2) to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P(2). Because cellular PI(4,5)P(2) is resynthesized rapidly, steady state PI(4,5)P(2) changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P(2) with Ci-VSP allows for the quantification of relative PI(4,5)P(2) affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K(+) channels to Ci-VSP allowed for comparison of PI(4,5)P(2) affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P(2) depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P(2) and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P(2) affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P(2). While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P(2) affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P(2) that differ in their accessibility to PLC and VSPs. Frontiers Media S.A. 2015-06-19 /pmc/articles/PMC4472987/ /pubmed/26150791 http://dx.doi.org/10.3389/fphar.2015.00127 Text en Copyright © 2015 Rjasanow, Leitner, Thallmair, Halaszovich and Oliver. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Pharmacology Rjasanow, Alexandra Leitner, Michael G. Thallmair, Veronika Halaszovich, Christian R. Oliver, Dominik Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility |
title | Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility |
title_full | Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility |
title_fullStr | Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility |
title_full_unstemmed | Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility |
title_short | Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P(2) affinity, phosphoinositide selectivity, and PI(4,5)P(2) pool accessibility |
title_sort | ion channel regulation by phosphoinositides analyzed with vsps—pi(4,5)p(2) affinity, phosphoinositide selectivity, and pi(4,5)p(2) pool accessibility |
topic | Pharmacology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472987/ https://www.ncbi.nlm.nih.gov/pubmed/26150791 http://dx.doi.org/10.3389/fphar.2015.00127 |
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