Redox properties of the calcium chelator Fura-2 in mimetic biomembranes.
Fura-2 is one of the most commonly used fluorescent dyes to analyze the cytosolic Ca(2+) concentration ([Ca(2+)](i)) of living cells. Fura-2-dependent measurements of [Ca(2+)](i) are susceptible to changes of pH, reactive oxygen species concentration and membrane potential. Fura-2 is often loaded over the lipophilic cell membrane into the cytosol of a cell in its esterified form (Fura-2/AM) which is then cleaved by endogenous esterases. We have analyzed the electrochemical properties of Fura-2/AM and Fura-2 salt by cyclic voltammetry ("three-phase" and "thin-film" electrode methods). Using Fura-2/AM as a redox facilitator, we were able to mimic the transport of various ions across a lipophilic barrier. We show that Fura-2/AM in this biomimetic set-up can be reversibly oxidized in a single electrochemical step. Its redox reaction was highly proton sensitive in buffers with pH< or =6. At physiological pH of around 7.0, the oxidation of Fura-2/AM was coupled to an uptake of mono-anions across the liquid-liquid interface. The voltage-dependence of the redox cycle was sensitive to the free Ca(2+) concentration, either after de-esterification of Fura-2/AM, or when Fura-2 salt was used. The complex between Fura-2 and Ca(2+) ions is ionic (complexation occurs via the dissociated negative groups of Fura forms), while the redox transformations in Fura-2 occurs at the nitrogen atoms of the amino groups. Our results suggest that redox transformations of the Fura-2 forms do not affect the binding ability toward Ca(2+) ions and thus do not interfere with [Ca(2+)](i) measurements.
Gulaboski
Rubin
R
CIQ-UP L4, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal.
Pereira
Carlos M
CM
Cordeiro
M Natalia D S
MN
Silva
A Fernando
AF
Hoth
Markus
M
Bogeski
Ivan
I
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004566:Electrodes / Q000592:standards; D016257:Fura-2 / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008567:Membranes, Artificial; D008954:Models, Biological; D015394:Molecular Structure; D010084:Oxidation-Reduction
D002614:Chelating Agents; D007477:Ions; D008563:Membrane Lipids; D008567:Membranes, Artificial; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2