Fig. 4

Modulation of SpSLC9C1 activity by cAMP. a Action of 1 mM cAMP in the pipette solution on the voltage dependence of SpSLC9C1 (V_1/2 = −53.4 mV). ([Na⁺]_o 0 mM; [Na⁺]_i 14 mM; pH_i = 7.2; pH_o = 7.4). b Normalized ΔR values were plotted against V_m to yield the V_1/2 values by a fit with the Boltzmann equation (w/o cNMP: V_1/2 = −70.9 ± 2.5 mV, s = 3.3 ± 0.9 mV, n = 7; cAMP: V_1/2 = −56.8 ± 2.7 mV, s = 4.6 ± 1.5 mV, n = 7; cGMP: V_1/2 = −67.8 ± 5.4 mV, s = 2.8 ± 1.0 mV, n = 9). c Flash photolysis of caged cAMP enhanced SpSLC9C1 activity at a holding voltage of −63 mV and saturated after 3–4 flashes, same conditions as in a. d Normalized ΔR values were plotted against the number of flashes. Black circles show values from c. Decreasing the light energy of the flash, saturation of SpSLC9C1 activity required 5–6 flashes (white circles). e The shift of V_1/2 and s values evoked by flash photolysis were similar to those using cAMP in the pipette (black triangles: V_1/2 = −55.3 ± 5.1 mV, s = 5.0 ± 0.6 mV, n = 3; for comparison: w/o cNMP (black circles) and 1 mM cAMP (white circles). f Replacing the Arg1053 in the CNBD by Gln (R1053Q) maintained a wild-type-like V_1/2 in the absence of cNMP (−70.8 ± 4.8 mV, s = 3.6 ± 1.1 mV, n = 5), but strongly reduced the V_1/2 shift by cAMP (−69.5 ± 3.8 mV, s = 3.9 ± 1.2 mV, n = 6). Mean values are summarized in Table 1