Background: Iron is an element necessary for all the organisms; its role in essential metabolism and cellular processes is fundamental for life. Studies on iron homeostasis, regulation and transport could be also done with a simplified model of professional phagocyte and pathogen host, Dyctiostelium discoideum (Dd). Aim: Characterize the different metal transport specificity and regulation of the two iron transporters of Dd, ddNramp1 and ddNramp2. Methods: Electrophysiological experiments with Two Electrode Voltage Clamp, X. laevis oocytes smoothies (XLOs) and immunolocalization experiments were performed to characterize these two divalent metal transporters and started with the synthesis of the two chimeric protein c-Nramp1 and c-Nramp2. Results: We demonstrated that c-Nramp1 is a sodium independent transporter with specificity for divalent transition metals, as mammal orthologs of SLC11 family, with maximal inward currents at acidic pH and a high affinity for substrate. We also detected a peculiar sodium leak current, mainly at neutral pH, that suggest the presence of a strong interaction in the binding site. We found that c-Nramp2 is electroneutral and transports divalent metal ions at all pH. Site directed mutagenesis on TMD6 restore the electrogenicity with small inward currents; XLOs analysis confirmed the electrophysiological results, these mutants are more functional than wild type c-Nramp2. Conclusions: Our findings on ddNramp1 contributed with novel insights on the mechanism of action of the mammalian ortholog, whereas similar studies on ddNramp2 suggested a role of the homologous proteins in protists, and helped to clarify the possible function of the contractile vacuole in divalent metal homeostasis.

Functional characterization of iron transporters Nramp1 and Nramp2 from Dictyostelium discoideum: a model of cellular iron homeostasis / Imperiali, Francesca Guia. - (2017).

Functional characterization of iron transporters Nramp1 and Nramp2 from Dictyostelium discoideum: a model of cellular iron homeostasis.

Imperiali, Francesca Guia
2017

Abstract

Background: Iron is an element necessary for all the organisms; its role in essential metabolism and cellular processes is fundamental for life. Studies on iron homeostasis, regulation and transport could be also done with a simplified model of professional phagocyte and pathogen host, Dyctiostelium discoideum (Dd). Aim: Characterize the different metal transport specificity and regulation of the two iron transporters of Dd, ddNramp1 and ddNramp2. Methods: Electrophysiological experiments with Two Electrode Voltage Clamp, X. laevis oocytes smoothies (XLOs) and immunolocalization experiments were performed to characterize these two divalent metal transporters and started with the synthesis of the two chimeric protein c-Nramp1 and c-Nramp2. Results: We demonstrated that c-Nramp1 is a sodium independent transporter with specificity for divalent transition metals, as mammal orthologs of SLC11 family, with maximal inward currents at acidic pH and a high affinity for substrate. We also detected a peculiar sodium leak current, mainly at neutral pH, that suggest the presence of a strong interaction in the binding site. We found that c-Nramp2 is electroneutral and transports divalent metal ions at all pH. Site directed mutagenesis on TMD6 restore the electrogenicity with small inward currents; XLOs analysis confirmed the electrophysiological results, these mutants are more functional than wild type c-Nramp2. Conclusions: Our findings on ddNramp1 contributed with novel insights on the mechanism of action of the mammalian ortholog, whereas similar studies on ddNramp2 suggested a role of the homologous proteins in protists, and helped to clarify the possible function of the contractile vacuole in divalent metal homeostasis.
Membrane transporter, electrophysiology, SLC11, cellular physiology, molecular physiology
Functional characterization of iron transporters Nramp1 and Nramp2 from Dictyostelium discoideum: a model of cellular iron homeostasis / Imperiali, Francesca Guia. - (2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11383/2090593
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