Asymmetric effects of amphipathic molecules on mechanosensitive channels

Abstract

Mechanosensitive (MS) ion channels are primary transducers of mechanical force into electrical and/or chemical intracellular signals. Many diverse MS channel families have been shown to respond to membrane forces. As a result of this intimate relationship with the membrane and proximal lipids, amphipathic compounds exert significant effects on the gating of MS channels. Here, we performed all-atom molecular dynamics (MD) simulations and employed patch-clamp recording to investigate the effect of two amphipaths, Fluorouracil (5-FU) a chemotherapy agent, and the anaesthetic trifluoroethanol (TFE) on structurally distinct mechanosensitive channels. We show that these amphipaths have a profound effect on the bilayer order parameter as well as transbilayer pressure profile. We used bacterial mechanosensitive channels (MscL/MscS) and a eukaryotic mechanosensitive channel (TREK-1) as force-from-lipids reporters and showed that these amphipaths have differential effects on these channels depending on the amphipaths' size and shape as well as which leaflet of the bilayer they incorporate into. 5-FU is more asymmetric in shape and size than TFE and does not penetrate as deep within the bilayer as TFE. Thereby, 5-FU has a more profound effect on the bilayer and channel activity than TFE at much lower concentrations. We postulate that asymmetric effects of amphipathic molecules on mechanosensitive membrane proteins through the bilayer represents a general regulatory mechanism for these proteins.

Figure 1

Schematics of 5-Fu and TFE molecules and their interaction with a POPE membrane (based on their equilibrium state). (A) timeline of the drug’s diffusion into lipid bilayer (B), and averaged depth of the penetration over the simulation time (C). Inset shows the adsorption of 5-FU (grey) into a POPE lipid bilayer (green) containing MscL (cyan). The phosphate atoms of the bilayer have been represented as grey spheres to better visualize the lipid-water interface. As it can be seen from (A), the 5-FU molecule is completely planner. The Topological Polar Surface Area of 5-FU is 58.2 Ų, while this value has been reported 20.2 Ų for TFE molecules. (C) The mean penetration depths of TFE and 5-FU molecules are − 3.02 ± 0.40 Å and − 1.47 ± 0.63, Å respectively (mean ± SD, the changes are statistically significant; P < 0.05; Student’s t-test was performed).

Figure 2

Effect of 5-FU and TFE adsorption to the bilayer from the cytoplasmic and periplasmic sides. Sn-1 order parameter (A, B, E, F) and Sn-2 order parameter (C, D, G, H) of a POPE bilayer containing MscL are affected by both amphipaths. Effect of the drugs on Sn-1 and Sn-2 when added to the cytoplasmic side (A–D) and periplasmic side (E–H). The vertical axes represent the absolute values of calculated deuterium order parameters which show that TFE molecules interact with the lipid molecules leading to an increase of the order parameters. While the presence of 5-FU increases the order parameter for carbon atoms located close to the lipid/water interface its presence decreases the order parameter for those in the bilayer center. Data represents mean ± SD of 3 replicate simulations using 2% v/v TFE and 25 mM 5-FU.

Figure 3

Structural rearrangement of MscL helices due to 5-FU and TFE insertion using MD simulations. Red arrows show the parts of the channel that are moving due to the presence of the drugs. Cyan WT (not treated), Grey is 5FU and Orange is TFE.

Figure 4

Effect of 5-FU and TFE on MscL conformation. Extracellular diameter of MscL increases when added 5-FU and TFE are added from the extracellular side. 5-FU exerted a larger effect on MscL conformation compared to TFE. Data represents mean ± SD of 3 replicate simulations.

Figure 5

Effect of DMSO, TFE or 5-FU on gating of G22S-MscL reconstituted into liposomes. (A)–(D) Representative current traces showing activation of G22S-MscL channels upon application of pressure ramps recording under different conditions with and without application of 5-FU of TFE. The black traces indicate currents recorded before application of a drug, while the red traces indicate currents recorded at 15 min after exposure of the liposome patch to drugs. (E–H) Boltzmann distribution functions determined in the absence and the presence of each amphipath. 0.1% DMSO was used to dissolve 5-FU in the recording solution. Notice the parallel left-shift in negative pressure required for MscL-G22S gating in the presence of both TFE or 5-FU, but not in the DMSO control experiments. (I)–(L) Line charts showing the mid-maximum pressure (P_1/2) of recordings in (A)–(D). Different from 0 min, for 0.1% DMSO, ns P = 0.9071, ns P = 0.8023, ns P = 0.9210 and n = 5; for 0.5% TFE, *P = 0.0177, **P = 0.0074, **P = 0.0011 and n = 5; for 250 µM of 5-FU, ***P = 0.0008, **P = 0.0029, **P = 0.0013 and n = 7; for 500 µM of 5-FU, **P = 0.0018, ***P = 0.0008, **P = 0.0044 and n = 6, at 5, 10 or 15 min after treatment. (M) Summary data of shifts of mid-maximum pressure. The shifts were calculated by subtracting mid-maximum pressure obtained at each time point from that obtained at 0 min. Different from DMSO control, for 0.5% TFE, *P = 0.0273, *P = 0.0133, **P = 0.0037 and n = 5; for 250 µM of 5-FU, **P = 0.0025, **P = 0.0058, **P = 0.0015 and n = 7; for 500 µM of 5-FU, **P = 0.0020, **P = 0.0010, **P = 0.0032 and n = 6, at 5, 10 or 15 min after treatment.

Figure 6

Differential effect of TFE on MscL and MscS expressed in MJF612 E. coli spheroplasts. (A) MscL and MscS are activated by application of a pressure ramp to an excised spheroplast patch (green and black trace). Addition of TFE from the cytoplasmic side reversibly abolishes MscS activity (blue trace showing only MscL activity). (B) Pressure ramps activating MscL channels in MJF612 spheroplast membranes pre and post addition of 2% v/v TFE. (C) Representative Boltzmann distribution functions of the MscL open probability in the absence and the presence of 0, 2 and 4% v/v TFE. (D) Quantification of the magnitude of the leftward shift in the P_1/2 of MscL after 2% v/v TFE addition and quantification of the absolute change in the P_1/2.

Figure 7

Effect of 5-FU on TREK-1 heterologously expressed in HEK293T cells. (A, B) 5-FU sensitizes TREK-1 from the cytoplasmic side (I/O), while it reduces the current from the opposite leaflet (O/O). (C) Normalized current before and after 5-FU, inside-out (left, n = 7, p = 0.0373) and outside-out (right, n = 8, p = 0.0462).

Figure 8

Diagram summarizing the effect of TFE and 5-FU on different types of MS channels. TFE (), sensitization (), desensitization ().