Figure 2.

Verification and further characterization in physiological solutions of a Ca²⁺-blocked current in RBCs of healthy adults. Whole cell patch clamp recordings in physiological (a K⁺-based internal and a Na⁺-based external) solutions. (A) Raw current traces from a representative RBC in an external solution containing 2 mM CaCl₂ at t₁ (dark blue), 0 mM CaCl₂ at t₂ (green), 2 mM CaCl₂ at t₃ (light blue), and 20 mM CaCl₂ at t₄ (violet). (B) I/V curves in 2 mM CaCl₂ (t₁) (dark blue), 0 mM CaCl₂ (t₂) (green), 2 mM CaCl₂ (2nd application) (t₃) (light blue), and 20 mM CaCl₂ (t₄) (violet)-external solutions (n = 7 (3) with n being the number of cells and in brackets the number of donors). (C) Bar chart of the normalized current at 100 mV with the successive application of 2 mM CaCl₂, 0 mM CaCl₂, 2 mM CaCl₂, and 20 mM CaCl₂-external solutions; colors match the conditions in A and B. (D) I/V curve of the Ca²⁺-blocked current (the current recorded in 2 mM CaCl₂-external solution at t1 was subtracted from the current recorded in 0 mM CaCl₂-external solution at t2 in physiological solutions). In order to determine the whole cell conductance based on the slope of the I/V curve (143 pS), the current is given in absolute values, which results, due to cell-to-cell variations, in bigger error bars (standard error of mean) compared with normalized currents. Currents were elicited by voltage steps from −100 to 100 mV for 500 milliseconds in 20 mV increments at V_h = −30 mV. Measurements were performed at room temperature. Data are presented as mean ± standard error of the mean. Significance is assessed with a paired Student t test and set at P < 0.05. Stars are used as follows: ∗∗ for P < 0.01, and ∗∗∗ for P < 0.001; n.s. stands for nonsignificant. RBC = red blood cell.