Inhibition of the endogenous volume-regulated anion channel (VRAC) in HEK293 cells by acidic di-aryl-ureas

The endogenous volume-regulated anion channel (VRAC) from HEK293 cells was pharmacologically characterized using the whole-cell patch-clamp technique. Under isotonic conditions a small (1.3 nS), Ca2+-independent Cl conductance was measured. However, swelling at 75% tonicity activated a VRAC identified as an outward-rectifying anion current (P-1 > P-Cl > P-gluconate), which was ATP-dependent and showed inactivation at positive potentials. Activation of this current followed a sigmoid time course, reaching a plateau conductance of 42.6 nS after 12-15 min (t(1/2) = 7 min). The pharmacology of this VRAC was investigated using standard Cl--channel blockers (NPPB, DIDS, and tamoxifen) as well as a new group (acidic di-aryl ureas) of Cl--channel blockers (NS1652, NS3623, NS3749, and NS3728). The acidic di-aryl ureas were originally synthezised for inhibition of the human erythrocyte Cl- conductance in vivo. NS3728 was the most potent VRAC blocker in this series (IC50 = 0.40 muM) and even more potent than tamoxifen (2.2 muM). NS3728 accelerated channel inactivation at positive potentials. These results show that acidic di-aryl ureas constitute a promising starting point for the synthesis of potent inhibitors of VRAC.