Lipid Affinity to the Voltage-Gated Potassium Channel KvAP

Abstract

Voltage-gated potassium channels (KV) are formed by a central conducting pore surrounded by four voltage sensor domains. Functional studies have revealed that biophysical properties of lipid molecules in the channels environment can have strong effects on the activity of KV channels. Here, we investigated the influence of different lipids as well as their affinity to KvAP channels. We carried out electrophysiology measurements by fusing vesicles containing purified channels into planar lipid bilayers with varied lipid compositions. We found that KvAP properties are mainly determined by the lipid composition in the vesicles and not the lipids in the bulk bilayer, suggesting that KvAP has a preferential affinity to the lipids it comes in contact with first. It also shows that there is very limited exchange of the annular lipids around the channels. Even with time and at higher temperature, lipids from the bilayer did not mix with those of the vesicles, suggesting a strong channel-lipid interaction or confinement of lipid molecules to a microdomain around the channel. The annular lipids in immediate proximity of the VSD determines the mid-activation point of KvAP. Additionally, a bulk effect from positively charged lipids forming the bilayer affected the slope of the conductance-voltage curves. Here, the energy barriers and thus the kinetic rates of pore opening and entering the inactivated state are affected. This effect could be neutralized by addition of counter charges to the bilayer. Our results suggest that the binding site proposed also for other Kv channels has a similar affinity for different phospho- and non-phospholipids whereas exchange with bulk lipids is very restricted. Apart from the structural information, this has also implications on the experimental design when working with membrane proteins reconstituted in planar lipid bilayer.