RESUMO
The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium+ (MPP+) and tetraethylammonium+ (TEA+) or inhibition of MPP+ uptake by the nontransported inhibitors tetrabutylammonium+ (TBuA+), tetrapentylammonium+ (TPeA+), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (Km) values, different IC50 values, and varying effects of mutations were determined. In addition, varying IC50 values for the inhibition of MPP+ uptake and varying effects of mutations were obtained when different MPP+ concentrations far below the apparent Km value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 µM MPP+ for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent Km values for TEA+ and/or MPP+ Mutation of Trp218 and Asp475 led to altered IC50 values for TBuA+, TPeA+, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC50 values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Catecolaminas/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Mutagênese/efeitos dos fármacos , 1-Metil-4-fenilpiridínio/metabolismo , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Mutagênese/fisiologia , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacologia , Ratos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia , Xenopus laevisRESUMO
In acidic, coal mining lake sediments, facultatively anaerobic Acidiphilium species are probably involved in the reduction of Fe(III). Previous results indicate that these bacteria can co-respire O2 and Fe(III). In this study, we investigated the capacity of the sediment microbiota to reduce Fe(III) in the presence of O2 at pH 3. In sediment microcosms with 4% O2 in the headspace, the concentration of Fe(II) increased at a rate of 1.03 micromol (g wet sediment)-1 day-1 within the first 7 days of incubation which was similar to the rate obtained with controls incubated under anoxic conditions. However, in microcosms incubated under air, Fe(II) was consumed after a lag phase of 8 h with a rate of 2.66 micromol (g wet sediment)-1 day-1. Acidiphilium cryptum JF-5, isolated from this sediment, reduced soluble Fe(III) with either 4 or 21% O2 in the headspace, and concomitantly consumed O2. However, the rate of Fe(II) formation normalized for cell density decreased under oxic conditions. Schwertmannite, the predominant Fe(III)-mineral of this sediment, was also reduced by A. cryptum JF-5 under oxic conditions. The rate of Fe(II) formation by A. cryptum JF-5 decreased after transfer from preincubation under air in medium lacking Fe(III). Acidiphilium cryptum JF-5 did not form Fe(II) when preincubated under air and transferred to anoxic medium containing Fe(III) and chloramphenicol, an inhibitor of protein synthesis. These results indicate that: (i) the reduction of Fe(III) can occur at low O2 concentrations in acidic sediments; (ii) Fe(II) can be oxidized at O2 concentrations near saturation; and (iii) the enzyme(s) responsible for the reduction of Fe(III) in A. cryptum JF-5 are not constitutive.