Probing S4 Re-Arrangement during Gating using Optical Tools

Abstract

Voltage Sensor Domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes in response to changes in the membrane potential. Voltage sensitivity relies on the transfer of gating charges located in the fourth transmembrane helix (S4) across the electric field. The gating charge transfer has been proposed to be associated with changes in the secondary structure of the S4 between alpha and 3-10 helix, yet this conformational transition has not been observed to date. Because such transitions are expected to extend the distance between the two ends of the S4, we sought to identify changes in the distance between genetically-encoded spectroscopic probes (a Lanthanide-Binding-Tag and a fluorescent mCherry) placed at the two ends of the S4 of the Ciona intestinalis Voltage-Sensitive Phosphatase (Ci-VSP) using LRET. We previously showed that the length of the S4 of Ci-VSP tagged with LBT and mCherry does not exhibit long-lived voltage-dependent changes and that it most likely exists as an alpha helix. We report here that short-lived changes of the S4 length in this construct could not be detected using time-resolved FRET, indicating the unlikeliness of transient alpha to 3-10 transitions. Since the recent x-ray structure of the NavAb, sodium channel from Arcobactor butzlen, displays its whole S4 in a 3-10 conformation, we substituted the S4 of Ci-VSP (from G216 to S236) by the S4 of NavAb (from F95 to Q115). As for Ci-VSP, our LRET results with this chimera show that the NavAb S4 in the Ci-VSP frame does not undergo long-lived alpha-3-10 transitions, suggesting that it most likely resides in an alpha conformation. Our results show that in both constructs, the voltage-sensor operation from resting to active does not require alpha to 3-10 transitions of the whole S4. Supported by GM030376 and U54GM087519.