Photoactivation of a Mechanosensitive Channel

Abstract

Optogenetics in the conventional sense, i.e. the use of engineered proteins that gain their light sensitivity from naturally abundant chromophores, represents an exciting means to trigger and control biological activity by light. As an alternate approach, photopharmacology controls biological activity with the help of synthetic photoswitches. Here, we used an azobenzene-derived lipid analogue to optically activate the transmembrane mechanosensitive channel MscL which responds to changes in the lateral pressure of the lipid bilayer. In this work, MscL has been reconstituted in nanodiscs, which provide a native-like environment to the protein and a physical constraint to membrane expansion. We characterized this photomechanical system by FTIR spectroscopy and assigned the vibrational bands of the light-induced FTIR difference spectra of the trans and cis states of the azobenzene photolipid by DFT calculations. Differences in the amide I range indicated reversible conformational changes in MscL as a direct consequence of light switching. With the mediation of nanodiscs, we inserted the transmembrane protein in a free standing photoswitchable lipid bilayer, where electrophysiological recordings confirmed that the ion channel could be set to one of its sub-conducting states upon light illumination. In conclusion, a novel approach is presented to photoactivate and control cellular processes as complex and intricate as gravitropism and turgor sensing in plants, contractility of the heart, as well as sensing pain, hearing, and touch in animals.

Keywords: AzoPC; FTIR spectroscopy; Langmuir film; MscL; biomembrane; electrophysiology; nanodiscs; photolipids.

FIGURE 1

Light switching of the AzoPC monolayer: LB trough lateral pressure changes induced by intermittent light switching (blue/UV) on a monolayer of AzoPC at the air-water interface.

FIGURE 2

Current traces of MscL activation. MscL reconstituted into a mixed lipid bilayer of 20% AzoPC and 80% DPhPC. Activation of MscL was achieved by blue-light illumination (λ = 455 nm) of cis-AzoPC which lead to spontaneous openings of the channel after a delay time of about 5 s. The voltage across the bilayer was set to +20 mV.

FIGURE 3

IR spectra of AzoPC compared to DSPC. IR absorption spectra of DSPC (black) and AzoPC in the trans (blue) and cis (orange) state are overlaid to contrast their vibrational differences. Blue-filled are the modes arising due to the azobenzene-modified lipid tail mostly characteristic of the trans state, orange-filled mostly of the cis state. The inset contains a chemical sketch of both lipid structures. Spectra over the full mid-IR (MIR) range are available in Supplementary Figure S7.

FIGURE 4

(A) FTIR difference spectra of nanodiscs supplemented with AzoPC in the presence of MscL (blue for the cis to trans switching, red for the trans to cis). Vertical dashed lines indicate marker bands for the isomerization of AzoPC. (B) Same as (A) but in the absence of MscL. (C) Double-difference spectra that reflect the response of MscL by subtracting difference spectra in the absence (B) from spectra in the presence of MscL (A). The box refers to the amide I range which is indicative for changes in the C=O peptide bond, here of MscL (Insets). A diagram of a nanodisc with (A) and without (B) MscL reconstituted. The ion channel is in orange, lipids are in white and the scaffold proteins in blue.