Abstract
Aquaporins and Rh proteins can function as gas (CO&sub2; and NH&sub3;) channels. The present study explores the urea, H&sub2;O, CO&sub2;, and NH&sub3; permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [¹&sup4;C]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% CO&sub2;/33 mM HCO&sub3;⁻ (pHS increase) or 0.5 mM NH&sub3;/NH&sub4;⁺ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H&sub2;O-injected control oocytes. UT-B expression had no effect on the CO&sub2;-induced ΔpHS but doubled the NH&sub3;-induced ΔpHS. Phloretin reduced UT-B-dependent urea uptake (Jurea*) by 45%, Pf* by 50%, and (- ΔpHS*)NH&sub3; by 70%. p-Chloromercuribenzene sulfonate reduced Jurea* by 25%, Pf* by 30%, and (ΔpHS*)NH&sub3; 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 H&sub2;O and NH&sub3; 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 H&sub2;O and NH&sub3; permeability. Our data confirm that UT-B has significant H&sub2;O permeability and for the first time demonstrate significant NH&sub3; permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH&sub3; and H&sub2;O pass through the three monomeric urea channels in UT-B.