The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion.
The Amt/Mep ammonium channels are trimers in which each monomer contains a long, narrow, hydrophobic pore. Whether the substrate conducted by these pores is NH(3) or NH(4)(+) remains controversial. Substitution of leucine for the highly conserved tryptophan 148 residue at the external opening to Escherichia coli AmtB pores allowed us to address this issue. A strain carrying AmtB(W148L) accumulates much larger amounts of both [(14)C]methylammonium and [(14)C]methylglutamine in a washed cell assay than a strain carrying wild-type AmtB. Accumulation of methylammonium occurs within seconds and appears to reflect channel conductance, whereas accumulation of methylglutamine, which depends on the ATP-dependent activity of glutamine synthetase, increases for many minutes. Concentration of methylammonium was most easily studied in strains that lack glutamine synthetase. It is eliminated by the protonophore carbonyl cyanide m-chlorophenyl hydrazone and is approximately 10-fold higher in the strain carrying AmtB(W148L) than wild-type AmtB. The results indicate that AmtB allows accumulation of CH(3)NH(3)(+) ion in response to the electrical potential across the membrane and that the rate of flux through AmtB(W148L) is approximately 10 times faster than through wild-type AmtB. We infer that both mutant and wild-type proteins also carry NH(4)(+). Contrary to our previous views, we assess that E. coli AmtB does not differ from plant Amt proteins in this regard; both carry ions. We address the role of W148 in decreasing the activity and increasing the selectivity of AmtB and the implications of our findings with respect to the function of Rh proteins, the only known homologues of Amt/Mep proteins.
Fong
Rebecca N
RN
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA.
Kim
Kwang-Seo
KS
Yoshihara
Corinne
C
Inwood
William B
WB
Kustu
Sydney
S
D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D050296:Microbial Viability; D009154:Mutation / Q000235:genetics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D007477:Ions; D008744:Methylamines; D014364:Tryptophan; C027451:methylamine; D008239:Lysine