Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA
Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA<sup>+</sup>) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that ser...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-01-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/2/490 |
| _version_ | 1797416183116333056 |
|---|---|
| author | Ana Marcela Giudici Clara Díaz-García Maria Lourdes Renart Ana Coutinho Manuel Prieto José M. González-Ros José Antonio Poveda |
| author_facet | Ana Marcela Giudici Clara Díaz-García Maria Lourdes Renart Ana Coutinho Manuel Prieto José M. González-Ros José Antonio Poveda |
| author_sort | Ana Marcela Giudici |
| collection | DOAJ |
| description | Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA<sup>+</sup>) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA<sup>+</sup> binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA<sup>+</sup> bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA<sup>+</sup>–KcsA complex exhibits a loss in the affinity towards permeant K<sup>+</sup> at pH 7.0, when the channel is in its closed state, but maintains the two sets of K<sup>+</sup> binding sites and the W67–W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA<sup>+</sup>–bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA. |
| first_indexed | 2024-03-09T05:59:30Z |
| format | Article |
| id | doaj.art-fc53f0ff61574ad2a715efa2a9be8086 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T05:59:30Z |
| publishDate | 2021-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-fc53f0ff61574ad2a715efa2a9be80862023-12-03T12:11:01ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-01-0122249010.3390/ijms22020490Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsAAna Marcela Giudici0Clara Díaz-García1Maria Lourdes Renart2Ana Coutinho3Manuel Prieto4José M. González-Ros5José Antonio Poveda6Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, E-03202 Elche, SpainInstitute for Bioengineering and Bioscience (IBB), Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, PortugalInstituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, E-03202 Elche, SpainInstitute for Bioengineering and Bioscience (IBB), Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, PortugalInstitute for Bioengineering and Bioscience (IBB), Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, PortugalInstituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, E-03202 Elche, SpainInstituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, E-03202 Elche, SpainAlkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA<sup>+</sup>) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA<sup>+</sup> binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA<sup>+</sup> bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA<sup>+</sup>–KcsA complex exhibits a loss in the affinity towards permeant K<sup>+</sup> at pH 7.0, when the channel is in its closed state, but maintains the two sets of K<sup>+</sup> binding sites and the W67–W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA<sup>+</sup>–bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.https://www.mdpi.com/1422-0067/22/2/490potassium channelstetraalkylammonium saltsprotein thermal stabilityhomo-FRETC-type inactivationbinding affinity |
| spellingShingle | Ana Marcela Giudici Clara Díaz-García Maria Lourdes Renart Ana Coutinho Manuel Prieto José M. González-Ros José Antonio Poveda Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA International Journal of Molecular Sciences potassium channels tetraalkylammonium salts protein thermal stability homo-FRET C-type inactivation binding affinity |
| title | Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA |
| title_full | Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA |
| title_fullStr | Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA |
| title_full_unstemmed | Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA |
| title_short | Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA |
| title_sort | tetraoctylammonium a long chain quaternary ammonium blocker promotes a noncollapsed resting like inactivated state in kcsa |
| topic | potassium channels tetraalkylammonium salts protein thermal stability homo-FRET C-type inactivation binding affinity |
| url | https://www.mdpi.com/1422-0067/22/2/490 |
| work_keys_str_mv | AT anamarcelagiudici tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa AT claradiazgarcia tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa AT marialourdesrenart tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa AT anacoutinho tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa AT manuelprieto tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa AT josemgonzalezros tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa AT joseantoniopoveda tetraoctylammoniumalongchainquaternaryammoniumblockerpromotesanoncollapsedrestinglikeinactivatedstateinkcsa |