The Entamoeba histolytica EhPgp5 (MDR-like) protein induces swelling of the trophozoites and alters chloride-dependent currents in Xenopus laevis oocytes.
Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents the multidrug resistant phenotype due to the expression of the E. histolytica P-glycoproteins EhPgpl and EhPgp5. Here, we studied the protein EhPgp5 encoded by the EhPgp5 gene in emetine-sensitive trophozoites transfected with the pEhNEOPgp5 plasmid carrying the EhPgp5 gene. The transfected trophozoites increased their drug resistance slightly, but became bigger and globular. To investigate other EhPgp5 functions further, we microinjected the EhPgp5 mRNA in Xenopus laevis oocytes. Microinjected oocytes expressed EhPgp5 protein in their membranes and exhibited an ion current not present in the control oocytes. The antisense EhPgp5AS transcript, co-injected with the EhPgp5 mRNA, abolished the exogenous current, showing its specificity. Exogenous current was outward during depolarizing pulses. Reduction of the extracellular Cl- concentration displayed a reversible decrease of the current amplitude. Niflumic acid, 4,4-diisothiocyanatostilbene-2, 2'-disulfonic acid, and other Cl- channel blockers abolished the exogenous current, which was poorly modified by verapamil and changes in osmolarity of the medium. Our results suggest that the EhPgp5 protein could function as a Cl- current inductor and as a coadjuvant factor to avoid drug accumulation in the cell.
Delgadillo
Dulce María
DM
Program of Molecular Biomedicine, CICATA IPN, Col. Irrigación, México, DF.
Pérez
D Guillermo
DG
Gómez
Consuelo
C
Ponce
Arturo
A
Paz
Francisco
F
Bañuelos
Cecilia
C
Mendoza
Leobardo
L
López
César
C
Orozco
Esther
E
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D018118:Chloride Channels / Q000187:drug effects*; D003001:Cloning, Molecular; D004594:Electrophysiology; D004748:Entamoeba histolytica / Q000737:chemistry / Q000235:genetics*; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D015800:Protozoan Proteins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014982:Xenopus laevis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018118:Chloride Channels; C459579:EhPgp5 protein, Entamoeba histolytica; D015800:Protozoan Proteins; D012333:RNA, Messenger