Abstract
The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts (https://doi.org/10.5281/zenodo.6511116). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2–3 d.
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Data availability
Raw data for Figs. 9 and 10 can be obtained from the corresponding author upon request and at https://github.com/jacopofrallicciardi/Data-Determining-small-molecule-permeations.
Code availability
All the MATLAB scripts used in this study are available at https://doi.org/10.5281/zenodo.6511116 (ref. 36).
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Acknowledgements
The research was funded by an ERC Advanced grant (ABCVolume; no. 670578) and the EU CoFund program ALERT. We thank C. Presutti for testing the protocol.
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B.P. and M.G. conceived the idea and developed the design guidelines; M.G. performed the initial fluorescence measurements and developed the mathematical model for the data processing; J.F. performed the majority of the experiments, developed the protocol further and improved the computational data processing; J.F. and B.P. wrote the manuscript. B.P. supervised, resourced and led the project.
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Key references using this protocol
Gabba, M. et al. Biophys. J. 118, 422–434 (2020): https://doi.org/10.1016/j.bpj.2019.11.3384
Gabba, M. & Poolman, B. Biophys. J. 118, 435–447 (2020): https://doi.org/10.1016/j.bpj.2019.11.3383
Frallicciardi, J. et al. Nat. Commun. 13, 1605 (2022): https://doi.org/10.1038/s41467-022-29272-x
Key data used in this protocol
Frallicciardi, J. et al. Nat. Commun. 13, 1605 (2022): https://doi.org/10.1038/s41467-022-29272-x
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Frallicciardi, J., Gabba, M. & Poolman, B. Determining small-molecule permeation through lipid membranes. Nat Protoc 17, 2620–2646 (2022). https://doi.org/10.1038/s41596-022-00734-2
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DOI: https://doi.org/10.1038/s41596-022-00734-2
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