PAVSD · Photo-activated voltage-sensing domain in voltage-sensing proteins
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2010-04-01 → 2012-03-31
- Финансиране от ЕС
- 161 900 €
- Участници
- 1
- Схема
- MC-IIF
Линиите свързват координатора с партньорите.
Накратко на български
Калиевите канали в протеините се модифицират с фоточувствителни групи, за да се управляват чрез светлина. Това помага за разбирането на молекулните движения при активирането на протеиновите комплекси.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Photo-activated voltage-sensing domain in voltage-sensing proteins.
The aim of this project was to develop a new photoswitchable light-activated voltage-sensing domain, by incorporating synthetic photoisomerisable groups into the S1-S4 segment of Shaker voltage-gated potassium channels. This development would confer light sensitivity to voltage-activated proteins and allow manipulating remotely the voltage sensor, helping to understand the molecular movements involved in activation and their modulation within protein complexes. Based on molecular and structural information (Kv1. 2/Kv2. 1 paddle chimera) available on Shaker we identified several positions in the outer regions of S4 in an open conformation and the S1, S2 or S3b segments where light-sensitive compounds could be introduced, in a way that photo isomerisation would interfere reversibly with the voltage sensor movements. At the selected positions, we introduced cysteine residues by mutagenesis, and evaluated their functional expression by means of gating charge vs. voltage relationship in a mammalian cell line or Xenopus oocytes. We also synthesized cysteine-reactive, photoisomerisable compounds to be conjugated specifically at the selected positions in the channel. After screening the stability in physiological solution of several reactive photoisomerisable compounds, and the functional expression level of a group of cysteine mutant channels, only two photoswitches and five cysteine mutants were available to perform reliable experiments. In this reduced set of conditions, the gating charge characteristics of the modified channels was displaced after conjugation in some cases, but no light-dependence was observed. This result indicates that either the reactive photoswitch or the modified residues had a limited accessibility for reaction, that the specific sites of modification did not allow effective alterations of the voltage sensor movement upon photoisomerisation, or that the structural assumptions made on the voltage sensor movements during gating were not correct
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Eukaryotic cells have voltage-gated ion channels that are directly activated by voltage but never by light. Voltage-gated ion channels contain a voltage-sensing domain (VSD) that responds to changes in membrane potential by gating the pore domain. VSD are functionally independent domains within canonical voltage-activated ion channels, but are also components of voltage-sensing enzymes and can also form ion channels. Several strategies have been used to enable light regulation of ion channels that are not intrinsically light sensitive and these channels have been successfully used to control neuronal activity (Gorostiza & Isacoff 2007). In the case of voltage-gated potassium (Kv) channels, photoswitchable molecules have been described but they specifically act as covalently tethered channel blockers (Horn et al., 2000). To date, photo-regulation of VSDs has not been reported. Photoswitchable VSD would constitute an exclusive independent structure to remotely control voltage-sensing proteins. We expect voltage-sensing proteins will be rapidly and reversibly activated by light, which has potential use as a noninvasive method to study motion charging shape of VSD in vitro and in vivo. This project aims to develop a new photoswitchable light-activated VSD by incorporating an azobenzene chromophore into the VSD of Shaker Kv channel. Although the X-ray crystallographic structures of prokaryotic and same as eukaryotic potassium channels were recently published, the position and the molecular movement of the voltage sensor in the activated or deactivated state are still controversial. As a long term aim of this project, this approach could be extended to the recently cloned and described VSD proteins such as Ci-VSP and Hv1 (Murata el al., 2005; Sasaki et al., 2006; Ramsey et al., 2006). Light activation of the voltage sensor would elucidate movements within this structure and report on the gating mechanisms of voltage-activated ion channels and VSD-containing proteins.
Оригинален текст от CORDIS (на английски).
Участници
- FUNDACIO INSTITUT DE BIOENGINYERIA DE CATALUNYA · BarcelonaКоординаторИспания
Връзки
Данни: CORDIS, © Европейски съюз