Fig. 3.

Calcium inhibition of the DMT1-mediated H⁺ leak and Fe²⁺-evoked currents. (A) Typical continuous current recordings at membrane potential (V_m) of 70mV in a control oocyte (i) and in an oocyte expressing DMT1 (ii). Oocytes were superfused with Ca²⁺-free medium at pH 7.5 for the periods shown by the empty bars, or with Ca²⁺-free medium at pH 5.5 (hatched bars). We presented 20 mM Ca²⁺ as indicated by the gray bars and 20 μM or 2 μM Fe²⁺ (black bars). (B) Ca²⁺-induced current (I^Ca) in the absence of Fe²⁺ metal ion as a function of Ca²⁺ concentration. Data were fit by equation 3 to describe the Ca²⁺ inhibition of the DMT1-mediated H⁺ leak current: I_max = 16.4 ± 1.0 nA, K_i^Ca = 1.1 ± 0.3 mM (adjusted r² = 0.88, P < 0.001). (C) Current-voltage relationships of the currents induced by 2 μM Fe²⁺ alone (Fe, filled symbols) or 2 μM Fe²⁺ plus 20 mM Ca²⁺ (Fe + Ca, empty symbols) (n = 4, paired). (D) Effect of intracellular Ca²⁺ chelation on the Ca²⁺ inhibition of the DMT1-mediated Fe²⁺-evoked current. We voltage-clamped at −70 mV four oocytes expressing DMT1, and measured currents evoked by 2 μM Fe²⁺ alone or 2 μM Fe²⁺ plus 20 mM Ca²⁺ before (−2 min) and after (10, 20, 30 min) microinjection with EGTA. Each symbol shape represents an individual oocyte. The mean Fe²⁺-evoked current at −2 min was −88 (SD 64) nA and did not change with time (one-way repeated measures ANOVA, P = 0.83). We obtained percent inhibition by Ca²⁺ and found no effects over time (one-way repeated measures ANOVA, P = 0.18).