Analysis of CorA-Catalyzed Mg2+ Selective Currents in Xenopus Oocytes

Abstract

In prokaryotes, Magnesium homeostasis is tightly controlled through the workings of three distinct transport systems, with CorA being the primary source of Mg2+ uptake. The recent structures of CorA from the thermophilic eubacteria Thermotoga maritima (TmCorA) have provided an excellent model for a molecular understanding of Mg2+ transport. Yet, a detailed characterization of CorA function has been conspicuously missing so far, as only in-vivo bulk assays have been available to evaluate Mg2+ translocation leaving unanswered fundamental questions related to CorA ion selectivity and specificity, gating mechanism and regulation. Here we have developed a system for high-level expression of TmCorA in Xenopus oocytes that allows for an accurate functional readout of Mg2+ transport through several electrophysiological techniques. Mg2+ currents from TmCorA in the order of 1 to 10 μA were routinely obtained with the cut-open voltage clamp technique and two electrodes Voltage clamp.We demonstrate that CorA act as a strong inward rectifier, an observation in agreement with its physiological role as the primary source of Mg2+ uptake. As expected, Mg2+ currents are blocked by Co(III) hexamine, a structural analog of a hydrated Mg2+. CorA selectivity towards Magnesium vs. other divalent and monovalents was characterized using a perfusion TEV setup. The divalent selectivity series for CorA was determined as Co2+>=Mg2+>Ni2+>Zn2+>>>Ca2+ = Ba2+. Macroscopic CorA currents record from oocytes macropatches show that Mg2+-binding to the cytoplasm domain act as a gating factor. Single channel transitions are still below our detection capabilities and efforts are ongoing to characterize the conductance of TmCorA through noise analysis. The ability to evaluate CorA function by means of electrophysiological methods will allow for a detailed analysis of structure/function relationships in CorA.