Final Report Summary - PAVSD (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