RESUMO
The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.
Assuntos
Antígenos HLA/genética , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Transferrina/metabolismo , Capacitância Elétrica , Antígenos HLA/metabolismo , Células HeLa , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ferro/metabolismo , Técnicas de Patch-Clamp , Receptores da Transferrina/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Since its introduction by Neher and Sakmann in 1976 the patch-clamp technique has been extensively used to study processes such as signalling and synaptic transmission, but also for monitoring endo- and exocytosis. Since biological membranes behave like electrical capacitors high-resolution measurements of membrane capacitance allow detection of small changes in membrane surface area that accompany exocytosis and endocytosis. We here describe our recent work on patch-clamp capacitance measurements in stably transfected HeLa cells expressing HFE, the hereditary hemochromatosis gene product, under the control of a tetracycline-sensitive promotor. By means of whole-cell and cell-attached techniques we were able to reveal transferrin-induced decreases in membrane capacitance reflecting increased endocytosis at the single cell level. Moreover, cell-attached recordings revealed significant alterations in the formation of single endocytic vesicles. Time-resolved measurements of cell membrane capacitance provide a new methodological approach to study the endocytic uptake of transferrin and its regulation by HFE, the hereditary hemochromatosis protein.