Intracellular lifetime in genetically modified yeast cells by FEXSY and PGSE - accounting for different intra/extracellular T₂

Water transport across the cell membrane is regulated by special proteins called aquaporins (AQP). There are different types of AQP in the human body and their ability to transport water varies. Yeast cells can be genetically modified to express human AQP instead of their own. One method that is commonly used to study water permeability of the membrane, which is closely related to the intracellular lifetime, is to subject the cells to a hyperosmotic shock and measure the cell shrinkage using light scattering [1].

In yeast, the intracellular lifetime can be determined non-invasively in equilibrium by using diffusion NMR methods such as filter-exchange spectroscopy (FEXSY) [2]. It has been shown that FEXSY can be used to measure the intracellular lifetime in baker's yeast, which is in the range of a few hundred milliseconds.

The T₂ relaxation times of the water in the genetically modified yeast cells are much shorter than those for baker's yeast. In addition, in genetically modified yeast, there are also significant differences in T₂ between the intra- and extracellular sites causing a bias in the estimation of the intracellular lifetime. To avoid this bias, it is important to account for the T₂ differences between the two exchanging sites.

Here we use a single-PGSE D-T₂ correlation experiment with constant diffusion time and variable echo time to estimate the difference in T₂ between the intra- and extracellular water. We also present a method to measure the intracellular lifetime using FEXSY with a double stimulated echo sequence and how to correct for T₂ differences. The two different methods are used in combination in order to account for varying intracellular lifetimes and T₂ in yeasts expressing different types of human AQP.