NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.
PLoS One
; 9(4): e93200, 2014.
Article
em En
| MEDLINE
| ID: mdl-24699187
ABSTRACT
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Membrana Celular
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Trocadores de Sódio-Hidrogênio
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Proteínas de Escherichia coli
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Escherichia coli
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Proteínas Mutantes
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Potenciais da Membrana
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Mutação
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article