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.

Structural Basis for Glycerol Efflux and Selectivity of Human Aquaporin 7

“…However, the water permeability of human AQP7 was recently measured in a proteopolymerosome assay in which AQP7 was shown to have similar water permeability as the orthodox aquaporins (Gotfryd et al, 2018). In a cellular environment glycerol is likely to be pre-sent, as opposed to the glycerol-free proteopolymerosome setup (Palmgren et al, 2017). Hence, these experimental results support the MD simulations presented here, showing reduced water permeability of AQP7 in the presence of glycerol.…”

“…The mainly hydrophobic environment of the extracellular vestibule accompanied by a lack of hydrogen-bonding partners for the hydroxyl groups of glycerol may facilitate glycerol release from the pore. Compared with the orthodox aquaporins, AQP7 has been shown to have significantly lower water permeability in cell-based assays (Katano et al, 2014b;Palmgren et al, 2017). However, the water permeability of human AQP7 was recently measured in a proteopolymerosome assay in which AQP7 was shown to have similar water permeability as the orthodox aquaporins (Gotfryd et al, 2018).…”

“…However, a recent structural and functional study of AQP10 suggests that AQP10 facilitates the transport of glycerol in a pHdependent manner in adipocytes (Gotfryd et al, 2018). In contrast, AQP7 has been shown to have the same glycerol activity at both high and low pH (pH 6-8) (Katano et al, 2014b;Palmgren et al, 2017) and to regulate glycerol efflux in adipocytes by a hormonally controlled trafficking mechanism, i.e., shuttling between the lipid droplet and the plasma membrane (Rodriguez et al, 2011). Explicitly, AQP7 binds to Perilipin 1 (PLIN1) under lipogenic conditions, which is a protein decorating the lipid droplet in adipocytes (Hansen et al, 2016;Londos et al, 1999).…”

“…The role of AQP7 as a glycerol efflux channel is well described in the literature; however, it is still debated whether it also facilitates the transport of water at similar rates as the orthodox aquaporins (strict water channels) (Kondo et al, 2002;Palmgren et al, 2017). Thus, to clarify the functional mechanism of human AQP7, we determined two X-ray structures of AQP7 at 1.9 and 2.2 Å resolution and performed molecular dynamics (MD) simulations of the glycerol passage through the pore of AQP7.…”

Involvement of aquaglyceroporins in energy metabolism in health and disease

“…Xenopus laevis oocytes or cells expressing rat, mouse or human AQP7 permeate water, as well as glycerol, urea and H 2 O 2 [50,56,57]. Compared to orthodox AQPs, AQP7 displays lower water permeability in cell-based assays [58,59] but similar water permeability in proteopolymersome assay performed using a glycerol-free set up [60], or reduced water permeability in the presence of glycerol using molecular dynamics simulations [61]. The AQP7 structure has been elucidated in recent works using X-ray crystallography and molecular-dynamics stimulations [57,61].…”