Movement of NH₃ through the human urea transporter B: a new gas channel

Am J Physiol Renal Physiol. 2013 Jun 15;304(12):F1447-57. doi: 10.1152/ajprenal.00609.2012. Epub 2013 Apr 3.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 4-Chloromercuribenzenesulfonate / pharmacology
  • Amino Acid Substitution
  • Ammonia / metabolism*
  • Animals
  • Carbon Dioxide / metabolism
  • Gases / metabolism*
  • Humans
  • Hydrogen-Ion Concentration
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Molecular Dynamics Simulation
  • Oocytes
  • Osmosis
  • Permeability / drug effects
  • Phloretin / pharmacology
  • Urea / metabolism*
  • Water / metabolism
  • Xenopus

Substances

  • Gases
  • Membrane Transport Proteins
  • urea transporter
  • Water
  • Carbon Dioxide
  • 4-Chloromercuribenzenesulfonate
  • Ammonia
  • Urea
  • Phloretin