Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins

Abstract

Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.

Keywords: Aquaglyceroporin; Aquaporin; NMR; P. pastoris; Water transport.

Fig. 1

A novel yeast strain of P. pastoris that is impermeable for water, glycerol, and arsenic trioxide. Comparison of solute permeation in different strains of P. pastoris. a Cells were subjected to a hyperosmotic shock causing outward movement of water. Normalized Light Scatter Intensity (NLSI) was plotted over time (s). The rate constant, k, was obtained by curve fitting using the equation: $y=A_1·e^{-k_1·t}+A_2$ (A ₁ Amplitude, k rate constant, t time, A ₂ = offset), and are shown as bars (inset), values are mean of duplicates, and error bars denote SD. b Glycerol accumulation per OD unit measured as a function of time after addition of [¹⁴C] glycerol. Values shown are mean normalized to the mean 0-value in each experiment and error bars denote standard deviation (SD). c Cells were spotted on Control media (BMMY, Buffered Methanol complex Media) with and without NaAsO₂ in 10 times dilution series to monitor As(III) sensitivity. A representative image is shown

Fig. 2

Quantification of aquaglyceroporins levels in the plasma membrane. a Quantitative Western blot analyses of aquaglyceroporins present in the plasma membrane. Protein content in plasma membrane fractions were analyzed by SDS-PAGE and Western blotting detecting Pma1 membrane marker and the polyhistidine-tag of the AQPs. Note that the typical aquaporin migrates in the gel as different mono/oligomers. b Quantification of the aquaglyceroporins present in the plasma membrane (His-signal divided by Pma1-signal), values are mean, and error bars denote SD

Fig. 3

Diffusion NMR method used to measure water exchange in cells. Water transport efficiency was measured for the different strains using the NMR diffusion technique. a–c Examples of results from NMR diffusion experiments showing normalized signal intensity for (a) aqy1Δagp1Δ + hAQP3, (b) aqy1Δagp1Δ + hAQP7, and (c) aqy1Δagp1Δ + hAQP9 as a function of diffusion weighting b. The upper graph shows signal attenuation curves from the PGSE experiment for increasing echo times, t _E, indicated by arrow. The lower graph shows signal attenuation curves from the FEXSY experiment where arrow indicates increasing mixing times, t _m. (d) Statistical significance was established by one-way ANOVA (F(4,10) = 11.27, p = 0,001), and Tukey’s multiple comparisons test rendered statistical significant differences (p < 0.01) exclusively between aqy1Δagp1Δ + hAQP1 and the other strains (aqy1Δagp1Δ, aqy1Δagp1Δ + hAQP3, aqy1Δagp1Δ + hAQP7, aqy1Δagp1Δ + hAQP9), but non-significant differences in all other comparisons. The aqy1Δagp1Δ + hAQP1 strain was analyzed at 0 °C together with aqy1Δagp1Δ control, while the other strains were analyzed at 20 °C. Values are mean values normalized to aqy1Δagp1Δ in each experiment (ki/ki_aqy1Δagp1Δ) and error bars denote SEM

Fig. 4

Applying SFM to measure cell shrinkage as indicative for water efflux. a Cells were subjected to a hyperosmotic shock causing outward movement of water. Normalized Light Scatter Intensity (NLSI) was plotted over time (s). b Water exchange rate constant (k) for aqy1Δagp1Δ, aqy1Δagp1Δ + hAQP3, aqy1Δagp1Δ + hAQP7, aqy1Δagp1Δ + hAQP9, values are mean of triplicates, and error bars denote SEM. Statistical significance was investigated with one-way ANOVA (F(3,8) = 14.38, p < 0.01), and Tukey’s multiple comparisons test rendered statistical significant difference (p < 0.05) between aqy1Δagp1Δ and aqy1Δagp1Δ + hAQP7 and non-significant differences comparing aqy1Δagp1Δ with aqy1Δagp1Δ + hAQP3 and aqy1Δagp1Δ + hAQP9

Fig. 5

The human aquaglyceroporin are efficient glycerol facilitators. Human aquaglyceroporins overexpressed in aqy1Δagp1Δ double-deletion strain were tested for their glycerol transport properties at two different pHs by supplementing a mix of glycerol and ¹⁴C glycerol. a At pH 7.5, cells were exposed to glycerol and the accumulation plotted at different time points. The relative glycerol uptake per OD unit is plotted (normalized to the mean 0-value in each experiment, and error bars shows SD). b Relative glycerol uptake per OD unit at 30 s (pH 7.5), values are mean of triplicates normalized to aqy1Δagp1Δ in each experiment, and error bars denote SEM. c Relative glycerol uptake per OD unit at 30 s (pH 6.0), values are mean of duplicates normalized to aqy1Δagp1Δ in each experiment, and error bars denote SD

Fig. 6

Human AQP7 and AQP9 are efficient As(III) facilitators. a Cells are spotted in 10-time dilution series on Control (BMMY, Buffered Methanol complex Media) and with NaAsO₂ supplemented. b Accumulated As(III) concentrations at t = 300 s the presence of 100 mM NaAsO₂ determined using ICP-MS. Values are mean of triplicates normalized to aqy1Δagp1Δ in each experiment, and error bars denote SEM