Influence of Lipid Bilayer Thickness on Ion Channels Using Single-Channel Voltage-Clamp Fluorescence Imaging

Abstract

The lipid molecules forming the cell membrane can strongly influence ion channel function dependent on several physical properties such as the electrostatic charge of the headgroup or the hydrophobic thickness. However, in contrast to other channel modulators such as voltage or ligands, the lipid membrane cannot be readily controlled. In order to modulate the surrounding lipid environment, the channels have to be purified and reconstituted into the desired lipid bilayer matrix. As these steps can prove challenging, the direct effect of lipids on ion channel function remains elusive and the underlying mechanisms poorly understood. Here, we inserted labeled KcsA (E71A) potassium channels into lipid bilayers of different thickness (length of carbon chain) made of either pure PC or a 3:1 mixture with a charged phospholipid (PG). To further identify and characterize their effect on channel activity, we measured simultaneously ionic current and position of single ion channels using a bilayer voltage-clamp system combined to a single-molecule fluorescence microscope. In all recordings containing several channels, clustering appeared to be important. Direct clustering events of freely diffusing single KcsA channels were observed in bilayers of various thicknesses, while channel activity remained constant. However, channel function appeared to be modulated by the bilayer thickness. Interestingly, despite a negative phospholipid headgroup has been previously suggested as an essential component for KcsA function, channel activity was still recorded without PG for bilayers close to physiological membrane thickness (∼27 A), suggesting that mechanical stress may compensate for the absence of the negatively charged head group. Moreover, clustering could also have a regulating effect since appearance of coupled channel activity showing significantly higher conductance than what is expected for KcsA was observed in the presence of clusters.