Login to view PhD Thesis

Enter your username and password here in order to log in on the website:


Forgot your password?

Ammonia transport in aquaporins

Lars Morten Holm

Summary

Aquaporins (AQPs) are membrane proteins known to mediate passive water transport. Several of them are also permeable to glycerol and other small hydrophilic molecules. They have been located to both plasma membranes as well as membranes of intracellular organelles.  

In the present thesis, I have demonstrated that AQPs 3, 7, 8, 9, and a plant AQP (TaTIP2;1) are permeable to NH3 and NH4+ by using an oocyte expression system in which volume changes and electrophysiological parameters were measured simultaneously. I found that: 1) NH4Cl-exposure of oocytes, which expressed ammonia-permeable AQPs, generated a clamp current, swelled the oocytes, and acidified extracellular pH; 2) formamide and methylammonia/methylammonium, which structurally resemble ammonia, permeated the ammonia-permeable AQPs as shown by uptake studies and measurements of reflection coefficients; 3) expression of AQP8 or TaTIP2;1 in a yeast strain, deficient of endogenous NH4+-transporters, complemented the yeast growth when the yeast was grown in ammonia containing media.  

The permeability of mannitol, urea, glycerol, acetamide, and formamide in AQPs 6, 7, 8, and 9 was determined by means of reflection coefficients and by uptake measurements of radio-labelled substrates. AQP6 was permeable to urea, glycerol, acetamide, and formamide when exposed to HgCl2. AQP7 was permeable to glycerol and formamide but not to urea as previously published. AQP8 was only permeable to formamide and water in contrast to AQP9 which transported a wide variety of substances.  

The molecular mechanism behind the selectivity was studied by mutations of AQP1 and TaTIP2;1. Substitutions in the aromatic/arginine region of the pore mouth of AQP1 led to permeability of larger substrates than water including ammonia. Additionally, I found 3 amino acid residues responsible for the ammonia permeability in TaTIP2;1.  

My results point to NH3-mediated NH4+-transport through AQPs 3, 7, 8, 9, and TaTIP2;1 and I propose that AQPs 3, 7, 8, and 9 serve physiological roles as ammonia channels. In the liver, this may play a role in amino acid metabolism and detoxification of NH4+ whereas in the kidney, the AQPs may participate in acid-base-homeostasis.