Native Function of the Bacterial Ion Channel SthK in a Sparsely Tethered Lipid Bilayer Membrane Architecture

Abstract

The plasma membrane protects the interiors of cells from their surroundings and also plays a critical role in communication, sensing, and nutrient import. As a result, the cell membrane and its constituents are among the most important drug targets. Studying the cell membrane and the processes it facilitates is therefore crucial, but it is a highly complex environment that is difficult to access experimentally. Various model membrane systems have been developed to provide an environment in which membrane proteins can be studied in isolation. Among them, tethered bilayer lipid membranes (tBLMs) are a promising model system providing a solvent-free membrane environment which can be prepared by self-assembly, is resistant to mechanical disturbances and has a high electrical resistance. tBLMs are therefore uniquely suitable to study ion channels and charge transport processes. However, ion channels are often large, complex, multimeric structures and their function requires a particular lipid environment. In this paper, we show that SthK, a bacterial cyclic nucleotide gated (CNG) ion channel that is strongly dependent on the surrounding lipid composition, functions normally when embedded into a sparsely tethered lipid bilayer. As SthK has been very well characterized in terms of structure and function, it is well-suited to demonstrate the utility of tethered membrane systems. A model membrane system suitable for studying CNG ion channels would be useful, as this type of ion channel performs a wide range of physiological functions in bacteria, plants, and mammals and is therefore of fundamental scientific interest as well as being highly relevant to medicine.

Figure 1

(A) Schematic of a sparsely tethered lipid bilayer on a gold support comprised of DPhyTL (red) mixed with mercaptoethanol (green) as a spacer molecule, electrode arrangement and equivalent circuit used to fit EIS data, if not stated otherwise. (B) Structures of DPhyTL and the phospholipids used to form the lipid bilayers.

Figure 2

Equivalent electrical circuits used to fit EIS data. R1: electrolyte resistance, R2: membrane resistance, R3: submembrane reservoir/spacer resistance, C1: membrane capacitance, CPE2: gold interface/submembrane reservoir capacitance.

Figure 3

(A) 2 × 2 μm AFM image of an stBLM comprised of the DOPC-POPG-CL lipid mixture containing several SthK ion channels. (B) Height profiles: profile 1 is the membrane only and profiles 2 and 3 cross an ion channel and a defect, respectively. (C) AFM image of a single SthK ion channel (top) and height profile of the ion channel (bottom). Additional AFM data are shown in the Supporting Information in Figure S2.

Figure 4

(A) Neutron scattering plot of SthK-stBLM comprised of the DOPC-POPG-CL lipid mixture (open symbols are experimental data, lines represent the fits) and (B) fitted SLD profile. The inset shows the approximate change of the SLD profile across the membrane. An additional data set is shown in the Supporting Information, Figure S3.

Figure 5

(A) Cyclic AMP response to SthK embedded a membrane comprised of DPPC only. (B) Cyclic AMP response to SthK embedded in a DPPC/CL membrane. (C) Cyclic AMP response to SthK embedded in a DOPC/POPG/CL membrane architecture. (D) Faster response of an SthK ion channel to the ligand at 37 °C.