Rapid outer pore movements after opening in a KV1 potassium channel are revealed by TMRM fluorescence from the S3-S4 linker, and modulated by extracellular potassium.

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker KV channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian KV1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of KV1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth, that is not seen in Shaker despite significant homology between it and KV1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and KV channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K+ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K+, and an IC50 of 29 mM K+o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K+, and further support the idea that outer pore motions occur rapidly after KV1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.