Correlating ion channel structure and function

Abstract

Recent developments in cryogenic electron microscopy (cryo-EM) led to an exponential increase in high-resolution structures of membrane proteins, and in particular ion channels. However, structures alone can only provide limited information about the workings of these proteins. In order to understand ion channel function and regulation in molecular detail, the obtained structural data need to be correlated to functional states of the same protein. Here, we describe several techniques that can be employed to study ion channel structure and function in vitro and under defined, similar conditions. Lipid nanodiscs provide a native-like environment for membrane proteins and have become a valuable tool in membrane protein structural biology and biophysics. Combined with liposome-based flux assays for the kinetic analysis of ion channel activity as well as electrophysiological recordings, researchers now have access to an array of experimental techniques allowing for detailed structure-function correlations using purified components. Two examples are presented where we put emphasis on the lipid environment and time-resolved techniques together with mutations and protein engineering to interpret structural data obtained from single particle cryo-EM on cyclic nucleotide-gated or Ca²⁺-gated K⁺ channels. Furthermore, we provide short protocols for all the assays used in our work so that others can adapt these techniques to their experimental needs. Comprehensive structure-function correlations are essential in order to pharmacologically target channelopathies.

Keywords: Cryo-electron microscopy; Ion channel; Lipid bilayers; MthK; Nanodisc; Radioactive uptake assay; Single-channel recording; SthK; Stopped-flow fluorescence assay.

Figure 1:. General workflow for structure-function studies.

Optimized expression and purification of ion channel proteins is followed by reconstituting pure, homogeneous protein into liposomes or nanodiscs under identical, defined conditions in order to correlate results.

Figure 2:. Ion channel reconstitution into lipid nanodiscs

A schematic representation of a membrane protein reconstituted into lipid nanodiscs. The MSP ring is shown in orange, the lipid bilayer in grey, and the protein (from PDB-6CJU) in blue. B Gel filtration analysis (Superose 6 10/300) of small-scale reconstitutions with different MP:MSP:lipid ratios (grey and blue) for SthK is shown together with SDS-PAGE analysis of the peaks 1 and 3.

Figure 3:. Flux assays

A Schematic overview of the ⁸⁶Rb⁺ uptake assay. The readout reports on the amount of radioactivity inside the liposomes at different times after exposure to ⁸⁶Rb⁺ containing uptake solution. B Schematic representation of ⁸⁶Rb⁺ uptake over time with increasing concentrations of agonist (from grey to black). C and D Mixing schemes for the single and the sequential mixing stopped-flow assay, respectively. E Principle of the fluorescence quenching. Fluorescent ANTS is shown in bright yellow, faded yellow represents quenched ANTS. F Representative data for an ion channel. The control in the absence of Tl⁺ (quencher) is shown in grey. In the presence of Tl⁺ but without agonist (blue) only very slow leakage of the quencher across the liposomal membrane is observed. In the presence of Tl⁺ and agonist (orange) rapid quenching reflecting channel activity is recorded. For each condition six technical repeats are shown in different shades of grey, blue, or orange, respectively. The ion channel representation (blue) in this figure was prepared from PDB-6CJU, the liposomal membrane is shown in rose and grey.

Figure 4:. Scheme of a horizontal bilayer setup

Proteo-liposomes are applied to the top chamber (left). After fusion of the liposomes with the bilayer and channel insertion (middle) the activity of a single ion channel can be recorded (right). Representative recordings at different voltages are shown for an engineered ion channel. The closed level (c) is indicated. PDB-6CJU was used for the channel representations (blue), liposomes or planar bilayers are shown in rose/gray.

Figure 5:. Structure and function of SthK

A Structures of apo SthK and cAMP-bound SthK (PDB: 6CJQ and 6CJU, respectively). B Single-channel recordings of SthK in 5:3:2 DOPC:POPG:Cardiolipin at +/− 20 mV and saturating cAMP concentration demonstrate the low open probability (Po) of SthK at voltages close to zero. Closed levels are indicated.

Figure 6:. Structure-function correlation for MthK channels

A Structures of MthK in different states (PDB IDs 6U6D, 6U5R, 6U68, 6U6E, 6U6H) with closed states in blue and open-inactivated states in red. B Schematic representation of the time-dependent activity of MthK as resolved in the sequential-mixing stopped-flow assay. Cryo-EM structures were assigned to functional states based on results as idealized in B.