FIG. 2.

KB-R7943 blocks the lobster SGC channel in a dose- and voltage-dependent manner. A: current-voltage (I-V) relationships determined from different patches recorded in control condition (gray trace) and in presence of 50 μM KB-R7943 (black trace) in different conditions of Ca²⁺ concentrations across the membrane. To generate I-V relationships, series of ramp protocols from −100 to +100 mV were applied. Whatever the internal and external Ca²⁺ concentrations, KB-R7943 appeared to strongly block the SGC current only at positive voltages. Slight reduction of the current seen at negative voltages in the presence of the drug seems to result from a short interval between the ramps and incomplete recovery of the channel activity after preceding inhibition. Note, when pipette was filled with PS the reversal potential of SGC current shifted by approximately +35 mV; corresponding corrections need to be done to directly compare voltage dependence of SGC channel conductance in different ion conditions. B: histogram of the averaged amplitude of the SGC current in the presence of KB-R7943 normalized to the SGC current in control at +100 mV from several patches in the same internal and external Ca²⁺ concentrations as in A. The blockage of the SGC current at positive voltages by KB-R7943 was strongly present in all Ca²⁺ conditions across the membrane tested. C: concentration dependence and voltage dependence of the blocking effect of KB-R7943 on the SGC channel. The percent inhibition of the averaged I-V curves for different concentrations of KB-R7943 was plotted as a function of the positive voltages. The percent inhibition was calculated as (mean current in control − mean current in presence of KB-R7943)/(current at +100 mV) × 100. D: concentration-inhibition curve for KB-R7943 on the mean amplitude of the SGC current at +100 mV. Averaged currents were normalized to the averaged current in absence of KB-R7943. Data points represent means ± SE of n = 3–11 patches and were fit to a Hill equation providing an IC₅₀ of 11.74 μM and a Hill coefficient 2.92. KB-R7943 blockage was reversible (n = 13, histogram). Mesylate (CH₃SO₃H, 200 μM) alone did not block the SGC channel (n = 2 both shown on the histogram). SN-6, a potent Na⁺/Ca²⁺ exchanger inhibitor, did not block the SGC current.