Structural determinants of NH3 and NH4+ transport by mouse Rhbg, a renal Rh glycoprotein.
Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH3/NH4+transport. The structure of Rhbg is not yet resolved; however, a high-resolution crystal structure of AmtB, a bacterial homolog of Rh, has been determined. We aligned the sequence of Rhbg to that of AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, histidine 183 (H183), histidine 342 (H342), and tryptophan 230 (W230), within the hydrophobic pore where they presumably serve to control NH3transport. A fourth residue, phenylalanine 128 (F128) was positioned at the upper vestibule, presumably contributing to recruitment of NH4+We generated three mutations each of H183, H342, W230, and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane, whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH3/NH4+and methyl amine (MA)/methyl ammonium (MA+)-induced currents, intracellular pH, and surface pH (pHs) among oocytes expressing the mutants, Rhbg, or injected with H2O. In H183 and W230 mutants, NH4+-induced current and intracellular acidification were inhibited compared with that of Rhbg, and MA-induced intracellular alkalinization was completely absent. Expression of H183A or W230A mutants inhibited NH3/NH4+- and MA/MA+-induced decrease in pHs to the level observed in H2O-injected oocytes. Mutations of F128 did not significantly affect transport of NH3or NH4+These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter.
Southeast Louisiana Veterans Health Care Network and Department of Medicine, Section of Nephrology, Department of Physiology, Tulane Hypertension and Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana.
Solange
S
Abdulnour-Nakhoul
Southeast Louisiana Veterans Health Care Network and Department of Medicine, Section of Nephrology, Department of Physiology, Tulane Hypertension and Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana.
Trang
T
Le
Southeast Louisiana Veterans Health Care Network and Department of Medicine, Section of Nephrology, Department of Physiology, Tulane Hypertension and Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana.
Edd
E
Rabon
Southeast Louisiana Veterans Health Care Network and Department of Medicine, Section of Nephrology, Department of Physiology, Tulane Hypertension and Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana.
L Lee
LL
Hamm
Southeast Louisiana Veterans Health Care Network and Department of Medicine, Section of Nephrology, Department of Physiology, Tulane Hypertension and Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana nakhoul@tulane.edu.
Nazih L
NL
Nakhoul
D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D014982:Xenopus laevis
D006023:Glycoproteins; D026901:Membrane Transport Proteins; C486033:RhBG protein, mouse; D000641:Ammonia
ammonia; electrophysiology; gene expression; ion-sensitive micro-electrodes; kidney; oocyte
Blood Rev. 2006 Mar;20(2):93-110
15961204
Anal Chem. 1981 Dec;53(14):2267-9
7316213
Am J Physiol Renal Physiol. 2010 Nov;299(5):F1065-77
20719974
Trends Biochem Sci. 1997 Dec;22(12):460-1
9433124
Am J Physiol Cell Physiol. 2015 Dec 1;309(11):C747-58
26354748
Proc Natl Acad Sci U S A. 2010 May 25;107(21):9638-43
20457942
Am J Physiol Cell Physiol. 2010 Sep;299(3):C695-705
20592240
Am J Physiol. 1984 Jul;247(1 Pt 2):F35-44
6742203
Pflugers Arch. 2006 Mar;451(6):701-7
16273393
J Biol Chem. 2001 Jan 12;276(2):1424-33
11024028
Contrib Nephrol. 1991;92:119-23
1756634
Gastroenterology. 2003 May;124(5):1432-40
12730882
J Biol Chem. 2004 Apr 16;279(16):15975-83
14761968
J Struct Biol. 2007 Aug;159(2):243-52
17660063
Mol Cell. 2006 Sep 1;23(5):663-71
16949363
Am J Physiol Renal Physiol. 2006 Feb;290(2):F297-305
16131648
Science. 2004 Sep 10;305(5690):1587-94
15361618
J Biol Chem. 2000 Aug 18;275(33):25641-51
10852913
Physiol Rev. 1989 Jan;69(1):179-249
2643123
Am J Physiol Cell Physiol. 2010 Dec;299(6):C1386-97
20810915
Am J Physiol Gastrointest Liver Physiol. 2005 May;288(5):G1036-47
15576624
Transfus Clin Biol. 2006 Mar-Apr;13(1-2):70-84
16584906
Contrib Nephrol. 1985;47:110-5
4064682
Am J Physiol Renal Physiol. 2005 Jan;288(1):F170-81
15353405
Reproduction. 2013 Jul 31;146(3):283-96
23904565
Mol Aspects Med. 2013 Apr-Jun;34(2-3):629-37
23506896
Nature. 2008 Nov 20;456(7220):339-43
19020613
Transfus Clin Biol. 2006 Mar-Apr;13(1-2):128-31
16580862
J Am Chem Soc. 2012 Jun 27;134(25):10419-27
22631217
Am J Physiol Renal Physiol. 2001 Jan;280(1):F1-9
11133509
J Biol Chem. 2006 Jul 14;281(28):19578-87
16672228
J Am Soc Nephrol. 2003 Mar;14(3):545-54
12595489
Transfus Clin Biol. 2006 Mar-Apr;13(1-2):65-9
16564194
Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17090-5
15563598
Am J Physiol Renal Physiol. 2001 Aug;281(2):F255-63
11457716
J Physiol. 2004 Sep 15;559(Pt 3):751-9
15284342
Nat Genet. 2000 Nov;26(3):341-4
11062476
Contrib Nephrol. 1985;47:125-9
4064684
Br J Haematol. 2005 Nov;131(4):543-51
16281947
Genetics. 2006 Aug;173(4):2005-19
16702425
Am J Physiol Renal Physiol. 2003 Feb;284(2):F323-37
12388412
Am J Physiol Renal Physiol. 2014 May 15;306(10):F1107-20
24647713
Proc Natl Acad Sci U S A. 2008 Apr 1;105(13):5040-5
18362341
Biochem J. 2005 Oct 1;391(Pt 1):33-40
15929723
Electrolyte Blood Press. 2009 Jun;7(1):14-9
21468180
Biochem Biophys Res Commun. 1997 Jun 9;235(1):103-7
9196044
Transfus Clin Biol. 2006 Mar-Apr;13(1-2):147-53
16580864