Abstract
Members of the Amt family of channels mediate the transport of ammonium. The form of ammonium, NH3 or NH4(+), carried by these proteins remains controversial, and the mechanism by which they select against K(+) ions is unclear. We describe here a set of Escherichia coli AmtB proteins carrying mutations at the conserved twin-histidine site within the conduction pore that have altered substrate specificity and now transport K(+). Subsequent work established that AmtB-mediated K(+) uptake occurred against a concentration gradient and was membrane potential-dependent. These findings indicate that the twin-histidine element serves as a filter to prevent K(+) conduction and strongly support the notion that Amt proteins transport cations (NH4(+) or, in mutant proteins, K(+)) rather than NH3 gas molecules through their conduction pores.
Highlights
The Amt family of ammonium channels does not conduct Kϩ
Our analysis shows that the twin-histidine element serves as a substrate selectivity filter and leads us to suggest that Amt channels utilize an electrogenic transport mechanism in which ammonium crosses the phenyl ring constriction as NH4ϩ, deprotonates and traverses the conduction pore in close association with its Hϩ, and reprotonates prior to entering the cytoplasm
Controversy remains over the form of ammonium carried by members of the Amt family
Summary
The Amt family of ammonium channels does not conduct Kϩ. Results: E. coli AmtB variants carrying certain mutations in the conserved twin-histidine element transport Kϩ against a concentration gradient. Subsequent work established that AmtB-mediated K؉ uptake occurred against a concentration gradient and was membrane potential-dependent These findings indicate that the twin-histidine element serves as a filter to prevent K؉ conduction and strongly support the notion that Amt proteins transport cations (NH4؉ or, in mutant proteins, K؉) rather than NH3 gas molecules through their conduction pores. Our analysis shows that the twin-histidine element serves as a substrate selectivity filter and leads us to suggest that Amt channels utilize an electrogenic transport mechanism in which ammonium crosses the phenyl ring constriction as NH4ϩ, deprotonates and traverses the conduction pore in close association with its Hϩ, and reprotonates prior to entering the cytoplasm
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