The biophysical and structural mechanisms underlying mechanosensitivity of the TREK-2 potassium channel

The mechanosensitive K2P K+ channels play important roles in a wide range of physiological processes, including touch, balance, pain, and hearing. The ability of these channels to sense changes in pressure within the cell membrane is essential to these processes. However, the structural and biophysical mechanisms underlying this remain unclear. In this thesis, multiscale molecular dynamics simulation techniques were used to investigate the biophysical and structural mechanisms underlying mechanosensitivity of K2P channels. First, how TREK-2 responded to membrane stretch was investigated by simulating TREK-2 embedded in a POPC bilayer subjected to negative pressure (i.e. positive tension). The results show that TREK-2 is intrinsically sensitive to changes in bilayer tension and that the channel switches conformation from the 'Down' state to the 'Up' state in response to membrane stretch. However, although the pressure profile changes symmetrically in both outer and inner leaflets, the structural response of TREK- 2 is highly asymmetric. The changes mainly occurred in the lower half of protein. TREK-2 was therefore next examined under asymmetric tension (i.e. tension only applied in one of two leaflets). Interestingly, TREK-2 only responded to positive tension in the inner leaflet indicating it has the ability to distinguish a variety of force directions within the membrane. Since, the mechanisms underlying mechanosensitivity and intracellular pH sensitivity are thought to be similar, I also examined the role of a critical mutation in the proximal C-terminus of TREK-1 (E306A). This revealed that E306A mimics the protonated state and changes the preferred environment of this residue from water to lipid and consequently assists movement up towards the bilayer. In addition, a new force analysis tool was developed to provide more insight into why some K2P channels are mechanosensitive and some are not. Overall, this thesis improves our understanding of the molecular mechanisms underlying the mechanosensitivity of K2P channels and ion channels in general.</p

The biophysical and structural mechanisms underlying mechanosensitivity of the TREK-2 potassium channel

The mechanosensitive K2P K+ channels play important roles in a wide range of physiological processes, including touch, balance, pain, and hearing. The ability of these channels to sense changes in pressure within the cell membrane is essential to these processes. However, the structural and biophysical mechanisms underlying this remain unclear. In this thesis, multiscale molecular dynamics simulation techniques were used to investigate the biophysical and structural mechanisms underlying mechanosensitivity of K2P channels. First, how TREK-2 responded to membrane stretch was investigated by simulating TREK-2 embedded in a POPC bilayer subjected to negative pressure (i.e. positive tension). The results show that TREK-2 is intrinsically sensitive to changes in bilayer tension and that the channel switches conformation from the 'Down' state to the 'Up' state in response to membrane stretch. However, although the pressure profile changes symmetrically in both outer and inner leaflets, the structural response of TREK- 2 is highly asymmetric. The changes mainly occurred in the lower half of protein. TREK-2 was therefore next examined under asymmetric tension (i.e. tension only applied in one of two leaflets). Interestingly, TREK-2 only responded to positive tension in the inner leaflet indicating it has the ability to distinguish a variety of force directions within the membrane. Since, the mechanisms underlying mechanosensitivity and intracellular pH sensitivity are thought to be similar, I also examined the role of a critical mutation in the proximal C-terminus of TREK-1 (E306A). This revealed that E306A mimics the protonated state and changes the preferred environment of this residue from water to lipid and consequently assists movement up towards the bilayer. In addition, a new force analysis tool was developed to provide more insight into why some K2P channels are mechanosensitive and some are not. Overall, this thesis improves our understanding of the molecular mechanisms underlying the mechanosensitivity of K2P channels and ion channels in general.</p

Role of cholesterol flip-flop in oxidized lipid bilayers.

We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0-50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol\u27s flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups

Fullerenes’ Interactions with Plasma Membranes: Insight from the MD Simulations

Understanding the interactions between carbon nanoparticles (CNPs) and biological membranes is critically important for applications of CNPs in biomedicine and toxicology. Due to the complexity and diversity of the systems, most molecular simulation studies have focused on the interactions of CNPs and single component bilayers. In this work, we performed coarse-grained molecular dynamic (CGMD) simulations to investigate the behaviors of fullerenes in the presence of multiple lipid components in the plasma membranes with varying fullerene concentrations. Our results reveal that fullerenes can spontaneously penetrate the plasma membrane. Interestingly, fullerenes prefer to locate themselves in the region of the highly unsaturated lipids that are enriched in the inner leaflet of the plasma membrane. This causes fullerene aggregation even at low concentrations. When increasing fullerene concentrations, the fullerene clusters grow, and budding may emerge at the inner leaflet of the plasma membrane. Our findings suggest by tuning the lipid composition, fullerenes can be loaded deeply inside the plasma membrane, which can be useful for designing drug carrier liposomes. Moreover, the mechanisms of how fullerenes perturb multicomponent cell membranes and how they directly enter the cell are proposed. These insights can help to determine fullerene toxicity in living cells

Bilayer Deformation, Pores, and Micellation Induced by Oxidized Lipids

The influence of different oxidized lipids on lipid bilayers was investigated with 16 individual 1 μs atomistic molecular dynamics (MD) simulations. Binary mixtures of lipid bilayers of 1-palmitoyl-2-linoleoyl-<i>sn</i>-glycero-3-phosphatidylcholine (PLPC) and its peroxide and aldehyde products were performed at different concentrations. In addition, an asymmetrical short chain lipid, 1-palmitoyl-2-decanoyl-<i>sn</i>-glycero-3-phosphatidylcholine (PDPC), was used to compare the effects of polar/apolar groups in the lipid tail on lipid bilayer. Although water defects occurred with both aldehyde and peroxide lipids, full pore formation was observed only for aldehyde lipids. At medium concentrations the pores were stable. At higher concentrations, however, the pores became unstable and micellation occurred. Data analysis shows that aldehyde lipids’ propensity for pore formation is due to their shorter and highly mobile tail. The highly polar peroxide lipids are stabilized by strong hydrogen bonds with interfacial water