Movement of NH₃ through 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.

Keywords: ammonia transport; carbon dioxide transport; membrane protein; urea transport; water transport.

Fig. 1.

Western blot of human urea transporter B (hUT-B) extracted from Xenopus oocyte plasma membranes. Left: molecular-mass (MW) markers are displayed. Middle and right: representative Western blots of detergent-solubilized, biotinylated protein extracts from oocyte lysates, isolated using NeutrAvidin Resin. Thus the bands in H₂O and UT-B represent hUT-B resident in the plasma membrane. Lanes 1 and 2 represent, respectively, the equivalent of 1 and 2 H₂O-injected oocytes. Lanes 3 and 4 represent, respectively, the equivalent of 1 and 2 oocytes expressing hUT-B.

Fig. 2.

Uptake of [¹⁴C]urea by oocytes injected with H₂O or cRNA encoding hUT-B. A: time course of [¹⁴C]urea uptake. Oocytes were incubated in 200 μl of ND96 containing 5 μCi of [¹⁴C]urea (1 mM total urea) and then removed after 0, 2, 5, and 10 min. Values represent individual oocytes from 1 batch of oocytes. B: uptake data at the 5-min time point. Values represent the means ± SE for individual oocytes from 3 different batches of oocytes studied on 3 different days as in A. Values represent the means and ± SE for individual oocytes. The number of oocytes for each time point are shown in parentheses. Error bars are omitted when smaller than the symbol. For statistical analysis, we performed a Student's t-test (two tailed).

Fig. 3.

Effect of inhibitors on [¹⁴C]urea uptake by oocytes injected with H₂O or cRNA encoding hUT-B. A: raw uptake data at the 5-min time point (see Fig. 2A). B: portion of uptake dependent on hUT-B. From the uptake of each hUT-B oocyte, we subtracted the mean uptake of day-matched H₂O controls. Data are means ± SE, with number of observations in parentheses. We performed a one-way ANOVA, followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons are show). All data were obtained on the same day, from the same batch of oocytes, exposed to urea and the mean H₂O data for 1 day was subtracted from everything else. Cont, control (H₂O-injected) oocytes; Phlor, exposed to phloretin; pCMBS, p-chloromercuribenzene sulfonate.

Fig. 4.

Osmotic water permeability (P_f) measurements from oocytes injected with H₂O or cRNA encoding hUT-B. Here we summarize data from 4 batches of oocytes, examined on 4 different days. Each day, we studied similar numbers of day-matched H₂O and hUT-B oocytes that we exposed to 100 mosmol/kgH₂O ND96 as we observed swelling. Values represent the means ± SE; the number of oocytes are in parentheses. We performed Student's t-test (two tailed) for statistical comparison.

Fig. 5.

Effect of inhibitors on the P_f of oocytes injected with H₂O or cRNA encoding hUT-B. A: we preincubated oocytes injected with H₂O or expressing hUT-B in a ND96 sham solution (Cont) or in ND96 containing 0.5 mM phloretin (Phlor) for 20 min or 1.0 mM pCMBS for 10 min. After washing the oocytes, we measured P_f as described in Fig. 4. B: bar graphs are corrected for the P_f of H₂O-injected oocytes and thus represents the UT-B dependent P_f. Each day we determined a mean P_f for H₂O-injected oocytes and subtracted this background value from the P_f of each UT-B oocyte studied on that day. We performed a one-way ANOVA, followed by a SNK analysis (P shown for individual comparisons).

Fig. 6.

Surface pH (pH_S) measurements in oocytes exposed to CO₂/HCO₃⁻ or NH₃/NH₄⁺. A, bottom: describe the basis for the observed changes in surface pH (pH_S) changes upon exposure to solutions containing 5% CO₂/33 mM/HCO₃⁻ or 0.5 mM NH₃/NH₄⁺. In each experiment, we exposed the oocyte to CO₂/HCO₃⁻ for 5–8 min (enough time for pH_S to spike upwards and then decay to a stable value), then washed out the CO₂/HCO₃⁻ for 10–15 min (enough time for pH_S to spike downwards and then decay to a stable values), and then finally exposed the oocyte to NH₃/NH₄⁺. Traces in A, top, are representative pH_S transients from oocytes injected with H₂O or expressing hUT-B. B: bars summarize the results of a larger number of experiments, like those shown in A. We performed Student's t-test (two tailed) for statistical comparison.

Fig. 7.

Effect of inhibitors on ΔpH_S in oocytes injected with H₂O or cRNA encoding hUT-B. A: we preincubated oocytes injected with H₂O or expressing UT-B in a ND96 sham solution (Cont) or in ND96 containing 0.5 mM phloretin (Phlor) for 20 min or 1.0 mM pCMBS for 10 min. After washing the oocytes, we assayed ΔpH_S during NH₃/NH₄⁺ exposures, as described in Fig. 6. B: bar graphs are corrected for the ΔpH_S of H₂O-injected oocytes, and thus represents the UT-B-dependent maximal ΔpH_S. Each day we determined a mean ΔpH_S for H₂O-injected oocytes and subtracted this background value from the ΔpH_S of each UT-B oocyte studied on that day. We performed a one-way ANOVA, followed by a SNK analysis (P shown for individual comparisons).

Fig. 8.

Structure of the selectivity filter and potential of mean force (PMF) profiles of NH₃ and H₂O permeation through the monomeric pore of bUT-B. A: key channel-lining residues around the selectivity filter forming the major substrate binding sites (S_i, S_m, and S_o) are shown in licorice. Residue numbers refer to bUT-B. B: PMF profile for H₂O calculated from unbiased simulations of wild-type bUT-B, double mutant (T172V/T334V), and single mutants (T172V and T334V). C: PMF profiles calculated using umbrella sampling simulations for permeation of NH₃ through monomeric pores, as describes in materials and methods.

Fig. 9.

Effect of point mutants T177V and T339V on [¹⁴C]urea, H₂O, and NH₃ influx. A: UT-B-dependent [¹⁴C]urea uptake data for oocytes injected with H₂O or expressing hUT-B, T177V, or T339V at the 5-min time point (similar to Fig. 2B). B: UT-B-dependent P_f values, as described in materials and methods, we exposed oocytes to 100 mosmol/kgH₂O ND96 as we observed swelling. C: UT-B-dependent changes in surface pH (pH_S) elicited by exposing oocytes to 0.5 mM NH₃/NH₄⁺. For each oocyte expressing hUT-B, T177V, or T339V, we obtained the raw parameter value {[¹⁴C]urea uptake, P_f, or (ΔpH_S)_NH3} and from that subtracted the corresponding parameter value of day-matched H₂O-injected controls; the result is the UT-B-dependent parameter value. In A–C, the values represent means ± SE for individual oocytes, from 3 different batches of oocytes studied on 3 different days. Number of oocytes is shown in parentheses. We performed a one-way ANOVA, followed by a SNK analysis (P shown for individual comparisons).