Abstract
The opening and closing of voltage-dependent ions channels depend upon conformational changes initiated in the voltage-sensors. The nature and extent of this rearrangement has been widely investigated in K+ channels, using various techniques, but so far, there is no agreement on this fundamental mechanism in K+-channel gating.We investigated the voltage-sensor operation using a LRET technique employing Tb3+ ions bound to several Lanthanide-Binding-Tags (LBT) genetically encoded at the top of the S3 and S4 segments of the KvAP channel. A fluorescently-labeled pore-blocking toxin, the Agitoxin-2, was conveniently used as an acceptor placed at a non-mobile position near the pore-axis. Three mutations, S179G, K181D and P176E, were systematically introduced in the pore region to increase toxin binding. Various fluorophores were covalently attached to cysteines individually inserted at positions N5, Q13 and D20 of the agitoxin. After purification, mutant channels were reconstituted into proteoliposomes and submitted to a Nernst-clamp procedure combining the use of valinomycin and a K+ chelator. This method allowed us to stably clamp the liposomal electrical potential at negative and positive potentials. Thus, this allowed us to stabilize the voltage-sensor in its closed state and in its open-inactivated state during the data acquisition period. Channels were then blocked with the fluorescent toxins and LRET measurements were recorded. Sensitized emissions of the acceptors were fitted with a square-base pyramidal multi-exponential model (Posson and Selvin, 2008) allowing the extraction of the four distances separating the position of the acceptor near the pore and the position of the donors located further away in the four subunits. The voltage-dependent coordinates of the LBTs indicate a rotational movement of the top of S4, with little participation of the top of the S3 segment.Support: FWF and AHA0725763Z, NIHGM068044 and GM30376.
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