RESUMEN
The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na(+) and K(+) conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/química , Secuencia de Aminoácidos , Animales , Aniones/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Femenino , Humanos , Litio/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos , Mutación Puntual , Potasio/química , Estructura Terciaria de Proteína , Sodio/química , Compuestos de Sulfhidrilo/química , Xenopus laevisRESUMEN
Previous results suggested that specific point mutations in human anion exchanger 1 (AE1) convert the electroneutral anion exchanger into a monovalent cation conductance. In the present study, the transport site for anion exchange and for the cation leak has been studied by cysteine scanning mutagenesis and sulfhydryl reagent chemistry. Moreover, the role of some highly conserved amino acids within members of the SLC4 family to which AE1 belongs has been assessed in AE1 transport properties. The results suggest that the same transport site within the AE1 spanning domain is involved in anion exchange or in cation transport. A functioning mechanism for this transport site is proposed according to transport properties of the different studied point mutations of AE1.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Sustitución de Aminoácidos , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Aniones/metabolismo , Cationes Monovalentes/metabolismo , Humanos , Transporte Iónico/fisiología , Mutación Missense , Mutación Puntual , Estructura Terciaria de Proteína , Xenopus laevisRESUMEN
Missense mutations in the erythroid band 3 protein (Anion Exchanger 1) have been associated with hereditary stomatocytosis. Features of cation leaky red cells combined with functional expression of the mutated protein led to the conclusion that the AE1 point mutations were responsible for Na(+) and K(+) leak through a conductive mechanism. A molecular mechanism explaining mutated AE1-linked stomatocytosis involves changes in AE1 transport properties that become leaky to Na(+) and K(+). However, another explanation suggests that point-mutated AE1 could regulate a cation leak through other transporters. This short paper intends to discuss these two alternatives.