RESUMEN
Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Beta vulgaris/metabolismo , Betaína/metabolismo , Proteínas Portadoras/metabolismo , Colina/metabolismo , Prolina/metabolismo , Amaranthus/genética , Amaranthus/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Sistemas de Transporte de Aminoácidos Neutros/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Atriplex/genética , Atriplex/metabolismo , Secuencia de Bases , Beta vulgaris/genética , Transporte Biológico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Floema/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ionóforos de Protónes/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Xilema/metabolismoRESUMEN
The mammalian proline transporter (PROT) is a high affinity Na(+)/Cl(-)-dependent transporter expressed in specific regions of the brain. It is homologous to other neurotransmitter transporters such as glycine, norepinephrine, serotonin, and dopamine transporters. PROT is enriched in glutamatergic synaptic terminals and may play an important role in the regulation of excitatory neurotransmission. No non-peptide small molecule inhibitors have been described for this transporter. To study its physiological role in the central nervous system and evaluate its potential as a therapeutic target, we established cell lines that stably express recombinant hPROT and characterized its kinetic properties for proline uptake. We then screened for inhibitors and identified a series of compounds that inhibit hPROT-mediated proline uptake. A known compound, benztropine, was found to inhibit hPROT with an IC(50) of 0.75microM. A series of novel compounds were also found, one of which, LP-403812, showed an IC(50) of approximately 0.1microM on both recombinant human and mouse PROT without significant inhibition of glycine and dopamine transporters at concentrations up to 10microM. This compound also inhibited proline transporter activity of mouse brain synaptosomes with the same potency. These inhibitors provide important tools for the understanding of PROT functions in the brain and may lead to the development of therapeutic agents for certain neurological disorders.