Fig. 3

Voltage-clamp pH_i fluorimetry of SpSLC9C1 activity. a Alkalinization induced by a 20 s step hyperpolarization to −100 mV using an inwardly directed Na⁺ gradient ([Na⁺]_i 14 mM; [Na⁺]_o 140 mM; pH_i = 7.2; pH_o = 7.4, forward mode, red). No pH_i change occurred in non-transfected CHO cells (black). b Acidification induced by a 20 s step hyperpolarization to −100 mV using an outwardly directed Na⁺ gradient ([Na⁺]_i 14 mM; [Na⁺]_o 0 mM; pH_i = 7.2; pH_o = 7.4, reverse-mode, blue). c Perfusion with symmetric solutions (black line) abolished the net Na⁺/H⁺ exchange due to a lacking gradient (pH_i = pH_o = 7.2, [Na]_i = [Na]_o = 14 mM). Voltage induced net Na⁺/H⁺ exchange was restored when the cell was perfused with 140 mM Na⁺ (pH_o = 7.4). d When Arg399 in T12 of the exchanger domain is replaced by Ala (R399A), Na⁺/H⁺ exchange was abolished. e pH_i responses to repetitive voltage steps from −40 to −100 mV in a CHO-SpSLC9C1 cell. Dotted line indicates resting pH_i. ([Na⁺]_i 14 mM; [Na⁺]_o 140 mM; pH_i = 7.2; pH_o = 7.4). f Repetitive stimulation of SpSLC9C1 activity (reverse mode) in CHO cells that co-express the H⁺-selective channel Hv1. Cells quickly recovered from acidification by activation of Hv1 at +47 mV. ([Na⁺]_i 14 mM; [Na⁺]_o 0 mM; pH_i = 7.2; pH_o = 7.4). g Voltage dependence of SpSLC9C1 activation was determined by stepping V_m between −23 and −103 mV to +47 mV (V_1/2 = −70.4 mV; [Na⁺]_i 14 mM; [Na⁺]_o 0 mM; pH_i = 7.2; pH_o = 7.4). h Normalized ΔR values were plotted against V_m to yield the V_1/2 values by a fit with the Boltzmann equation (V_1/2 = −70.9 ± 2.5 mV, s = 3.3 ± 0.9 mV, n = 7). Mean values are summarized in Table 1