Mechanical properties of lipid bilayers and regulation of mechanosensitive function: from biological to biomimetic channels

Abstract

Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.

Figure 1. The effect of LPC on the phase transition (change in heat capacity) of DPPC MLVs as measured by differential scanning calorimetry (DSC). (A) DPPC thermograms in the absence (0%) or presence of 10, 20 or 25 mol% LPC at the indicated temperatures. Arrows show the displacement of the pretransition temperature (T_p) which defines the ripple phase (P_β') in pure DPPC. A new pretransition appears at lower temperatures (probably indicating an interdigitated gel phase, L_βI), then it vanishes at high doses of LPC. In (B), the structure factor for cylindrical DPPC (S_i = 1.11) and inverted cone-shaped LPC (S_i = 0.78) are schematized (Lipid structures taken from ref. 40).

Figure 2. Multiple alignments between homologs of MscL channel from Bacteria, Archaea and Eukaryotes. Bold letters represent residues forming the lipid-exposed TM2, which is important for mechanosensitivity. Thin letters represent poor conserved residues in the periplasmic loop (P-loop) and into the first part of the C-terminal domain. Red letters show hydrophobic residues whereas black letters show hydrophilic ones. Brown bold letters are flanking Tyr or Trp residues at the periplasmic face of the membrane. Blue bold letters represent flanking Lys or Arg residues facing to the cytosol. Snorkeling side chains attached to the TM2 are shown also in blue with a positive charge. Residues showing a high degree of conservation are highlighted in yellow. Trp (W) is highlighted in orange and represents a poor conservation of this residue at this place (~10%).

Figure 3. Hydrophobicity plot for MscL channel homologs from E. coli (EcoMscL), Mycobacterium tuberculosis (TbMscL), the archaeon Methanosarcina barkeri (MthMscL), MscS encompassing TM1 (residues 22–61 from EcoMscS), as well as the α-PFPs: melittin, fragment α6 from Bid and α5 from Bax proteins (see text). The hydrophobicity plots were developed by the algorithm of Kyte and Doolittle (ref. 82) using a window of 9 and aligning residues according to the lipid-exposed EcoMscL-TM2 profile. The topology of EcoMscL protein is included indicating only TM segments and the P-loop. As in Figure 2, snorkeling side chains attached to the TM1 and TM2 are shown in blue with a positive charge. Position 75 (E. coli numbering) is occupied by a Tyr or, less frequently, a Trp and are shown as a rigid brown lateral chain. The inserted 3D helix shown corresponds to a monomer of melittin (PDB: 2MLT).

Figure 4. (A) Structure of the mechanosensitive channel MscL (PDB 2OAR, taken from ref. 9). (B) Helical wheel representation for the TM fragments of MscL and a monomer of melittin. Melittin and TM2 were modeled in α-helical wheel representations; since TM1 is larger, it was modeled as a π-helix, which promotes a better lipid exposing face. Hydrophobicity factors (H) obtained are: 0.817 (TM1); 0.959 (TM2) and melittin (0.511). Net charge (z): –1 (TM1); 0 (TM2); + 5 (melittin). Residues with hydrophobic side chains are shown in yellow. Small residues Gly and Ala are represented in gray and positively charged snorkeling residues, in blue. N- and C-ends are shown. Arrows show the hydrophobic moment. (C) Osmotically induced inflow of NH₄⁺ through melittin pores in MLVs of DOPC:DPPC (2:1). MLVs were concentrated at 30 mM in choline chloride. Melittin was added to a P/L ratio of 1/40 in the presence and absence of a hypoosmotic downshock. For the osmotic shock, the sample was diluted 1:70 into an ammonium solution (100 mM). Permeability was determined by the swelling rate for a period of time, measuring the change in turbidity at Abs = 450nm. All flux measurements were performed at 25°C. (D) Schematic drawing of a toroidal pore in a lipid bilayer, taken from ref. .