Unassisted Transport of Block Tryptophan through DOPC Membrane: Experiment and Simulation

Abstract

The passive transport of small molecules across the plasma membrane is a key physiological process. Literature measurements of membrane permeability to small molecules have varied widely. We used confocal microscopy to image the transport of molecules into a giant unilamellar lipid vesicle (GUV). Fluorescent dyes were used to trace the transport of molecules. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short exposure. We used this system to study the passive transport of carboxylic acids, which have many properties common to small-molecule drugs. The transport of these acids across cell membranes has been widely studied, but there is much variation in the reported permeabilities. By using pH-sensitive fluorescein-dextran to track the acids permeating through the GUV membrane, our results showed that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive precise permeability values. The permeabilities change with the same trend as oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules. We used the imaging technique described above to study the transport of protons across compositionally asymmetric lipid bilayers. Synthetic asymmetric GUVs were prepared via a microfluidic multiphase droplet flow technology to mimic membrane charge asymmetry. Negatively charged phosphatidylserine was added to an asolectin GUV on either the internal or external leaflet. The permeation rates of protons into and out of these GUVs were measured. The proton distribution across the asymmetric GUV membrane at equilibrium was also studied. This research can reveal how asymmetric cell membrane composition affects small molecule transport behavior in physiological processes.

Partition of Positively and Negatively Charged Tryptophan Ions in Membranes with Inverted Phospholipid Heads: Simulations and Experiments

“…9,15,16,33,34 Even the permeation of polar species occurs with distortion of the bilayer structure of the membrane. [35][36][37] In the calculations of the PMF (Figure 2), large local perturbations are observed when the charged permeants are close to the membrane center (Figure 8). The membrane is distorted with some phosphates moving to the membrane interior for W+ (Figure 8(a)) and choline groups getting closer to W-(Figure 8(b)).…”

Two Relations to Estimate Membrane Permeability Using Milestoning

“…The MFPT ⟨ t ⟩ of a diffusional encounter can be generally expressed as here V is the total volume of the system in question, m is the total number of isolated sinks, and A i is the total area of the i th sink. In our 1D case, the expression is simplified Taking the integral of eq and reversing the order of integration in the numerator, we get Noting the equivalence of ∫ b a ∫ z a f ( x )­d x d z = ∫ b a ∫ b z f ( x )­d x d z for symmetrical functions over z | b < z < a and dividing by J ( a ) = − A 1 we get The MFPT can be related to the permeability coefficient by integrating eq by parts where the second term on the RHS has been shown to be equal to the MFPT by others ,,, and can be derived by applying Fubini’s theorem. Rewriting in terms of P and ⟨ t ⟩ and rearranging we get the permeability as it relates to ⟨ t ⟩ This is formally equivalent to the ISD and obviates the calculation of the local diffusivity, requiring only the local PMF (or equivalently, the local stationary concentration of the permeant) and the MFPT.…”

“…Previously, milestoning has been applied to the permeability problem by Cardenas et al, studying the permeation of block tryptophan across a membrane. 27 Cardenas et al later studied the permeation of small peptide NATA across a DOPC bilayer using milestoning. In their later work, they proposed a scheme to estimate the permeability coefficient.…”

“…Salient descriptions of milestoning theory can be found in refs − . Previously, milestoning has been applied to the permeability problem by Cardenas et al, studying the permeation of block tryptophan across a membrane . Cardenas et al later studied the permeation of small peptide NATA across a DOPC bilayer using milestoning.…”

Physics-Based Method for Modeling Passive Membrane Permeability and Translocation Pathways of Bioactive Molecules

“…5 Several general physics-based computational methods have been applied to overcome these problems. All-atom molecular dynamics (MD) simulations, 31,[34][35][36][37][38][39][40][41] multiscale (CG/MD) simulations, 42,43 Monte Carlo simulations, 44 and simulations with milestoning algorithms 45,46 were used to obtain detailed information on the dynamics of small molecules in phospholipid bilayers. MD simulations in explicit lipid bilayers were used to calculate free-energy profiles of small molecules in membranes and their permeability coefficients, [35][36][37][38][39][40][41][42][43] evaluate their optimal orientations in the bilayer, 47 and predict BBB-permeable drugs.…”