Movement of NH₃through the human urea transporter B: a new gas channel

Abstract

Aquaporins and Rh proteins can function as gas (CO₂ and NH₃) channels. The present study explores the urea, H₂O, CO₂, and NH₃ permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [¹⁴C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (ΔpHS) caused by exposing oocytes to 5% CO₂/33 mM HCO₃⁻ (pHS increase) or 0.5 mM NH₃/NH₄⁺ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H₂O-injected control oocytes. UT-B expression had no effect on the CO₂-induced ΔpHS but doubled the NH₃-induced ΔpHS. Phloretin reduced UT-B-dependent urea uptake (Jurea*) by 45%, Pf* by 50%, and (- ΔpHS*)NH₃ by 70%. p-Chloromercuribenzene sulfonate reduced Jurea* by 25%, Pf* by 30%, and (ΔpHS*)NH₃ by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H₂O and NH₃ and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H₂O and NH₃ permeability. Our data confirm that UT-B has significant H₂O permeability and for the first time demonstrate significant NH₃ permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH₃ and H₂O pass through the three monomeric urea channels in UT-B.

Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel

“…pH i and pHS measurements. Our approach for measuring intracellular (pHi) and surface pH (pHS)was the same as previously described (9,10,32,33). Briefly, oocytes under a dissecting microscope were impaled with both pH i and Vm microelectrodes, and a pHS electrode with a tip diameter of ϳ20 m was placed near the cell surface (see below).…”

“…Ϫ , we determined the maximum size of the pHS transient (i.e., the "spike height" or ⌬pHS) as previously described (9,10,(32)(33)(34). Briefly, we calibrated the pHS electrode before applying CO2-HCO 3 Ϫ (in ND96) by pulling the electrode away from the oocyte and taking a voltage measurement in the bulk extracellular fluid (assumed pH, 7.50).…”

“…Moreover, at least in plants, AQPs appear to play an important physiological role in CO 2 transport (12,50,51). In addition to the aquaporins, other protein families transport gas, including the Rh proteins, which conduct NH 3 and CO 2 (6,25,33,43), and most recently the UT urea transporters, which conduct NH 3 but not CO 2 (9).…”

Computing membrane-AQP5-phosphatidylserine binding affinities with hybrid steered molecular dynamics approach

“…(Krane et al, 2001a, Krane et al, 2001b, Verkman, 2002, Burghardt et al, 2006, Delporte and Steinfeld, 2006, Horsefield et al, 2008, Törnroth-Horsefield et al, 2010, Gravelle et al, 2013, Janosi and Ceccarelli, 2013, Zhang and Chen, 2013, Eckhard et al, 2014) It is also clear from in vitro experiments that AQP5 conducts transport of gas molecules as well,(Musa-Aziz et al, 2009, Geyer et al, 2013b, Qin and Boron, 2013) while carbon dioxide transport across the cell membrane is subject to much active debate. (Hub and de Groot, 2006, Wang et al, 2007, Missner et al, 2008a, Missner et al, 2008b, Hub et al, 2010, Wang et al, 2010, Wang and Tajkhorshid, 2010, de Groot and Hub, 2011, Itel et al, 2012, Kaldenhoff, 2012, Geyer et al, 2013a, Geyer et al, 2013c, Endeward et al, 2014, Hulikova and Swietach, 2014, Kaldenhoff et al, 2014) However, how AQP5 facilitates the transport of apolar, hydrophobic gas molecules is currently subject to much discussion. (Wang et al, 2007, Kaldenhoff et al, 2014) The X-ray structure of the AQP5 crystal tells us that AQP5 is tetrameric in conformation and that the four monomers in quasi-four-fold symmetry leave a significant void in the middle along the symmetry axis.…”

Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

“…The reason for such differences in sensitivity to NH 4 Cl are not clear. They may be related to the possible expression of ammonium transport systems 25,26 . Similarly the sodium-free solution that is used for rinsing the loading solution may not be tolerated by cells, such as cardiomyocytes for example.…”