Determining the Target of Membrane Sterols on the Gating of Voltage-Gated Potassium Channels using Voltage-Clamp Fluorometry

Abstract

Membrane lipids can affect the gating of voltage sensitive ion channels through different non-specific and specific mechanisms. It has been shown that cholesterol and 7-dehydrocholesterol (7DHC) have remarkable effects on the gating of Kv1.3 (shift in voltage-dependency of activation and a slower rate of activation) but it is not clear whether these effects are mediated by the actions through the voltage sensor domain (VSD) or the pore domain (PD). Our aim was to investigate whether the major target of the action of these membrane sterols is the VSD, PD or the coupling between these two domains. To test the specificity of the effect we carried out our measurements on channels with differing structures and gating mechanisms: Kv1.3 and the non-domain-swapped Kv10.1. Current recordings were performed with VCF technique using a Xenopus laevis oocyte expression system. To monitor the movement of the VSDs of the channels the specific cysteine residues on the S3-S4 extracellular linker were labeled with MTS-TAMRA. By electrophysiological measurements we determined typical current parameters of the channels and F-V curves from the fluorescent signal in control and sterol-loaded cells. In the oocyte expression system we were able to successfully reproduce the shift in voltage-dependency of activation and a slower rate of activation in the case of Kv1.3 and as a novel finding we obtained similar results with Kv10.1. We found no voltage shifts in the F-V curves paralleling the G-V shift in either ion channel, only the slopes of these curves were decreased in both cases. These results suggest that cholesterol and 7-DHC exert their effects by acting directly on the PD and/or the coupling mechanism, instead of influencing the VSD. Support: KTIA_NAP_13-2-2015-0009